Application of sirt5 gene inhibitor in preparation of drug for treating esophageal squamous cell carcinoma and interfering molecule

Abstract

The application relates to the technical field of medicines, and particularly discloses SIRT5 Application of a gene inhibitor in preparation of a drug for treating esophageal squamous cell carcinoma and an interfering molecule. SIRT5 The application finds that a functional gene related to the proliferation of esophageal squamous cell carcinoma is located between 13584111 bp and 13611862 bp of human chromosome 6, and the gene can be used as a target point for treating esophageal squamous cell carcinoma. SIRT5 The effective component of the gene inhibitor is SIRT5 shRNA of the gene, the gene inhibitor can inhibit the proliferation rate of cancer cells, and can be used for preparing a drug for treating esophageal squamous cell carcinoma.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to... SIRT5 Application of gene inhibitors in the preparation of drugs for the treatment of esophageal squamous cell carcinoma and interfering molecules. Background Technology

[0002] Human esophageal squamous cell carcinoma is a complex process involving the interaction of multiple genes and their products. Identifying key genes in the development and progression of human esophageal squamous cell carcinoma will help elucidate its intrinsic molecular biological mechanisms and will have significant implications for its prevention, diagnosis, and treatment. The search for key genes in human esophageal squamous cell carcinoma depends not only on the discovery of related novel genes but also on the continuous deepening of research into the functions of existing genes.

[0003] Therefore, it is necessary to explore new applications of existing genes in the treatment of human esophageal squamous cell carcinoma. Summary of the Invention

[0004] To explore new applications of existing genes in the treatment of human esophageal squamous cell carcinoma, this invention provides... SIRT5 Application of gene inhibitors in the preparation of drugs for treating esophageal squamous cell carcinoma and interfering molecules. This invention provides... SIRT5 The active ingredient of gene inhibitors is SIRT5 This invention utilizes shRNA, which is ligated into the GV248 vector and transformed into competent cells. Positive clones are screened to obtain interfering plasmids. A lentiviral three-plasmid packaging system is used to co-transfect the interfering plasmid and helper plasmid into 293T cells. The supernatant is collected, purified, and concentrated to obtain lentivirus. The lentivirus is then used to infect cells to inhibit [the growth of the gene]. SIRT5 Gene expression significantly inhibits the proliferation of esophageal squamous cell carcinoma cells.

[0005] This invention provides SIRT5 Application of gene inhibitors in the preparation of drugs for the treatment of esophageal squamous cell carcinoma.

[0006] The present invention provides SIRT5 Gene inhibitors can effectively silence genes. SIRT5 The gene significantly inhibited the proliferation of esophageal squamous cell carcinoma cells.

[0007] Furthermore, the drug is used to inhibit the proliferation of esophageal squamous cell carcinoma cancer cells.

[0008] Furthermore, the aforementioned SIRT5 Gene inhibitors SIRT5 The gene-interference molecule is the only effective component.

[0009] Furthermore, the aforementioned SIRT5The interference molecular target sequences of the gene are SIRT5-RNAi (668632-1) shown in SEQ ID NO.5, SIRT5-RNAi (668633-1) shown in SEQ ID NO.6, or SIRT5-RNAi (668634-1) shown in SEQ ID NO.7.

[0010] Furthermore, the interfering molecule targeting SIRT5-RNAi (668632-1) comprises the sense strand shown in SEQ ID NO.8 and the antisense strand shown in SEQ ID NO.9; The interfering molecule targeting SIRT5-RNAi (668633-1) contains the sense strand shown in SEQ ID NO.10 and the antisense strand shown in SEQ ID NO.11; The interfering molecule targeting SIRT5-RNAi (668634-1) contains the sense strand shown in SEQ ID NO.12 and the antisense strand shown in SEQ ID NO.13.

[0011] The present invention also provides an interfering molecule comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand are connected by a stem-loop structure to form a hairpin structure; The sense strand of the interfering molecule is shown in SEQ ID NO.8, and the antisense strand is shown in SEQ ID NO.9; Alternatively, the sense strand of the interfering molecule is as shown in SEQ ID NO.10, and the antisense strand is as shown in SEQ ID NO.11; Alternatively, the sense strand of the interfering molecule is shown in SEQ ID NO.12, and the antisense strand is shown in SEQ ID NO.13.

[0012] The present invention also provides SIRT5 Gene inhibitors, the SIRT5 The gene inhibitor is a lentivirus containing the aforementioned interfering molecule.

[0013] Furthermore, the lentivirus is constructed from the interfering molecule into the GV248 vector, transformed into competent cells, interfering plasmids are screened, and the supernatant is collected and purified and concentrated after transfection into 293T cells through a lentivirus three-plasmid packaging system.

[0014] The present invention also provides the aforementioned interfering molecule or the aforementioned... SIRT5 Application of gene inhibitors in the preparation of inhibitors for esophageal squamous cell carcinoma cells.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention identifies functional genes associated with the proliferation of esophageal squamous cell carcinoma through bioinformatics screening. SIRT5 Located between 13584111 bp and 13611862 bp on human chromosome 6; verified by immunohistochemistry, RT-qPCR and other experiments, it was found that... SIRT5 The gene is significantly overexpressed in esophageal squamous cell carcinoma tissues and cancer cell lines, and silencing the gene can significantly inhibit cancer cell proliferation, indicating that the gene can serve as a therapeutic target for esophageal squamous cell carcinoma.

[0016] This invention will... SIRT5 After the shRNA of the gene was ligated into the GV248 vector, it was transformed into competent cells. Positive clones were screened to obtain the interference plasmid. The interference plasmid and helper plasmid were co-transfected into 293T cells using a lentiviral three-plasmid packaging system. The supernatant was collected, purified, and concentrated to obtain lentivirus. The lentivirus was used to infect cells to inhibit [the virus / organization]. SIRT5 The gene expression of lentiviruses has a high infectivity and significantly inhibits the proliferation of esophageal squamous cell carcinoma cells, providing a new approach for the treatment of esophageal squamous cell carcinoma. Attached Figure Description

[0017] To more clearly illustrate the embodiments of the present invention or the existing solutions, the drawings used in the embodiments or the existing description will be briefly introduced below. Obviously, the 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.

[0018] Figure 1 for SIRT5 Gene expression in esophageal squamous cell carcinoma tissue and normal esophageal squamous epithelium; In the diagram, A is... SIRT5 Expression levels of the gene in esophageal squamous cell carcinoma tissue and normal squamous epithelium of the esophagus; B is SIRT5 Significant difference analysis of gene expression levels between esophageal squamous cell carcinoma tissue and normal esophageal squamous epithelium.

[0019] Figure 2 for SIRT5 Gene expression in cancer cell lines and normal esophageal mucosal squamous epithelial cells.

[0020] Figure 3 For carrier spectrum; In the figure, A represents the lentiviral vector GV248. B is a map of the virus packaging helper plasmid (Helper 1.0); C represents the map of the viral packaging helper plasmid (Helper 2.0).

[0021] Figure 4The images are cell images 72 hours after lentiviral infection. The naming rules for the infected images are as follows: CON empty cells, Sh-NC negative control, Sh-SIRT5#1, Sh-SIRT5#2 and Sh-SIRT5#3 are experimental group names, "1" is the first group of repeated infection, "2" is the second group of repeated infection, W is the bright field, and G is the green fluorescent field.

[0022] Figure 5 To analyze the expression of SIRT5 in cells after lentiviral infection; In the diagram, A represents the cell. SIRT5 Gene expression analysis; B represents the expression analysis of SIRT5 protein in cells.

[0023] Figure 6 The effect of SIRT5 gene lentivirus on the proliferation of esophageal squamous cell carcinoma cells was detected using CCK-8 assay. Detailed Implementation

[0024] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0025] Example 1: SIRT5 Gene screening and tissue expression analysis.

[0026] one, SIRT5 Gene screening This invention selects cancer group and adjacent normal group data from RNA-seq expression data of TCGA esophageal cancer data (TCGA-STAD) as research objects. Ballgown is used to compare the two groups of data to obtain significance p-values, q-values, and fold-changes between genes for differential gene screening. The screening criteria are as follows: P With values ​​<0.05, q <0.05, and Foldchange >2, functional genes associated with the proliferation of esophageal squamous cell carcinoma were successfully screened. SIRT5 The NCBI number is NM_001303627, located between 13584111 bp and 13611862 bp on human chromosome 6.

[0027] II. Expression of SIRT5 in esophageal squamous cell carcinoma tissue and normal esophageal squamous epithelium 1. Prepare paraffin sections of human esophageal squamous cell carcinoma tissue and normal esophageal squamous epithelial tissue, and dewax them with xylene, specifically xylene I, II and III for 15 min each, followed by benzene removal with a gradient of alcohols (100% alcohol for 10 min, 95% alcohol for 5 min, 90% alcohol for 3 min, and 85% alcohol for 1 min), and rinse with running water.

[0028] 2. According to the antibody instructions, select antigen retrieval, boil the Tris-EDTA alkaline retrieval solution in a pressure cooker, place the tissue section in the retrieval solution, boil for 20 minutes, and then allow it to cool naturally at room temperature.

[0029] 3. Wash 3 times with PBS, 5 min each time; place the tissue sections in 3% hydrogen peroxide solution and incubate at room temperature in the dark for 15 min to inactivate endogenous peroxidase; wash 3 times with PBS, 5 min each time; block with 5% BSA at room temperature for 30 min; add the diluted primary antibody to the tissue and incubate at room temperature for 2 h; wash 3 times with PBS, 5 min each time; add the reaction enhancement solution from the EnVisionplus kit to each tissue section and incubate at room temperature for 20 min; wash 3 times with PBS, 5 min each time; add the enzyme-labeled anti-mouse / rabbit IgG polymer from the EnVisionplus kit to each tissue section and incubate at room temperature for 30 min; wash 3 times with PBS, 5 min each time.

[0030] 4. As per the instructions, add 50 μl each of DAB-developing solutions 1, 2, and 3 to 850 μl of distilled water, mix well, and then drop the mixture onto the tissue section. Develop for 3 min, then rinse with tap water for 1 min to stop the process.

[0031] 5. Apply hematoxylin for 30 seconds, rinse with running water, differentiate with 1% hydrochloric acid alcohol for 5 seconds, rinse with running water, and then apply ammonia solution for 10 seconds to restore blue color, followed by rinsing with running water.

[0032] 6. Dehydrate the slide by applying 85% alcohol for 1 min, 90% alcohol for 3 min, 95% alcohol for 5 min, and 100% alcohol I and II for 10 min each; clear the slide by applying xylene I and II for 15 min each; then seal the slide with neutral resin and coverslip.

[0033] 7. Observe tissue sections under a microscope, collect images, and use ImageJ software to analyze the expression level of SIRT5 in esophageal squamous cell carcinoma tissue and normal esophageal squamous epithelial tissue.

[0034] The above experimental results (see) Figure 1 (A and B) indicate that SIRT5 expression levels in esophageal squamous cell carcinoma tissues are higher than in normal esophageal squamous epithelial tissues.

[0035] Example 2: SIRT5 Gene expression in cancer cell lines and normal esophageal mucosal epithelial cells.

[0036] Internal reference gene selection GAPDH (See Table 1) For SIRT5 Primers were designed for the gene sequence (see Table 2). Detection was performed using RT-qPCR. SIRT5 Expression of genes in normal human esophageal mucosal epithelial cells HEEC and human esophageal squamous cell carcinoma (ESCC) cells TE10, ECA109, KYSE450, and KYSE150.

[0037] Table 1. Primer information for internal reference genes Table 2 Primer sequences for the target gene I. Total RNA extraction (using the Takara RNAiso Plus kit, instruction manual link: https: / / www.takarabiomed.com.cn / DownLoad / 9108-9109.pdf) 1. The steps for extracting total RNA from adherent cells are as follows: Prepare 75% ethanol (prepared with DEPC water) and store at 4 ℃. Remove the cell culture flask, discard the original culture medium, wash the cells twice with sterile PBS, drain the liquid as much as possible, add 1 ml of RNAiso Plus, incubate at room temperature for 5 min, pipette tip-free, transfer to 1.5 ml RNase-free EP tubes, add 200 μl of chloroform to each tube, shake vigorously, incubate at room temperature for 5 min, centrifuge at 12000 rpm for 20 min, aspirate the supernatant, transfer to RNase-free EP tubes, add an equal volume of n-butanol, shake gently, incubate at room temperature for 20 min, centrifuge at 12000 rpm for 15 min, discard the supernatant, drain the liquid as much as possible, add 1 ml of 75% ethanol, incubate at room temperature for 5 min, centrifuge at 12000 rpm for 20 min, discard the supernatant, a small amount of white precipitate will be visible at the bottom of the tube, air dry in a fume hood, add 60 μl of DEPC water, mix thoroughly, use immediately or store at -80 ℃.

[0038] II. Preparation of cDNA (follow the TaKaRa kit instructions, protocol link: https: / / www.Takara biomed.com.cn / DownLoad / RR092S_RR092A.pdf).

[0039] Total RNA was quantitatively extracted using an ELISA reader, and its concentration was calculated. The reverse transcription reaction system (20 μl system) was prepared in an ice bath: 5× PrimeScript RT Master Mix and Total RNA were added to the PCR tube according to the proportions in Table 3, and RNase-Free H2O was added to a total volume of 20 μl. After mixing, the tube was briefly centrifuged.

[0040] The above system was reacted at 37 °C for 15 min, and then the RT enzyme was inactivated at 85 °C for 5 s. The resulting reverse transcription product cDNA was stored at -20 °C for later use.

[0041] Table 3 Reverse transcription reaction system 3. Perform RT-qPCR (follow the TaKaRa kit instructions).

[0042] Prepare the reaction system (20 μl system) according to the proportions in Table 4. Reaction conditions: 95 ℃ for 30 sec; 95 ℃ for 5 sec, 60 ℃ for 30 sec, 40 cycles; 95 ℃ for 15 sec, 60 ℃ for 1 h, 95 ℃ for 5 sec (detect the dissolution curve); use F = 2. -ΔΔCt Relative quantitative data analysis was performed (ΔCt = target gene Ct value - internal reference gene Ct value; -ΔCt = average ΔCt value of HEEC group - ΔCt value of ESCC group; 2) -ΔΔCt This reflects the relative expression level of the target gene in the ESCC group compared to the HEEC group.

[0043] Table 4 RT-qPCR reaction system The above experimental results (see) Figure 2 )show, SIRT5 The gene expression level in cancer cell lines was higher than that in normal esophageal mucosal epithelial cells.

[0044] Example 3: Application of lentivirus in the preparation of drugs for treating esophageal squamous cell carcinoma.

[0045] I. Construction of Lentivirals 1. SIRT5 Preparation of interfering molecules against SIRT5 Three RNA interference target sequences were designed (see Table 5), and then shRNA interference sequences were designed (see Table 6).

[0046] Table 5 SIRT5 RNA interference target sequence of gene Table 6 SIRT5 shRNA interference sequence of the gene Based on the shRNA interference sequence, shRNA oligonucleotide chains were synthesized. Specifically, SIRT5-RNAi(668632-1)-a and SIRT5-RNAi(668632-1)-b synthesized the oligonucleotide chain SIRT5-RNAi(668632-1); SIRT5-RNAi(668633-1)-a and SIRT5-RNAi(668633-1)-b synthesized the oligonucleotide chain SIRT5-RNAi(668633-1); and SIRT5-RNAi(668634-1)-a and SIRT5-RNAi(668634-1)-b synthesized the oligonucleotide chain SIRT5-RNAi(668634-1).

[0047] 2. Lentiviral vector acquisition Vector name: GV248 (this vector was provided by Genomics), vector as follows Figure 3 As shown in A.

[0048] Component sequence: hU6-MCS-Ubiquitin-EGFP-IRES-puromycin.

[0049] Reference number: CON208.

[0050] The reference insertion sequence is: TTCTCCGAACGTGTCACGT.

[0051] 3. Preparation of SIRT5-RNAi interference plasmid SIRT5-RNAi (668633-1), SIRT5-RNAi (668634-1), and SIRT5-RNAi (668632-1) oligonucleotide chains were ligated into the GV248 vector using the T4 ligation method to obtain SIRT5-RNAi interference plasmids. The SIRT5-RNAi interference plasmids include SIRT5-RNAi (668633-1), SIRT5-RNAi (668634-1), and SIRT5-RNAi (668632-1) interference plasmids.

[0052] The obtained SIRT5-RNAi interference plasmid was mixed into competent cells, and the bacterial culture was evenly spread on a plate. After incubation in an inverted state for 16 h, single colonies were picked for PCR identification. The identified positive clones were inoculated into LB liquid medium and incubated at 37 ℃ for 16 h. An appropriate amount of bacterial culture was sequenced. Plasmids were extracted and purified from bacterial cultures whose sequencing results were consistent with the target gene sequence. The purified SIRT5-RNAi interference plasmid was used for subsequent experimental procedures.

[0053] 4. Lentiviral packaging A lentiviral three-plasmid packaging system was used to package the virus with helper plasmids (Helper 1.0 and Helper 2.0). Figure 3 The B and C plasmids and the SIRT5-RNAi interference plasmid prepared above were co-transfected into 293T cells, and cell culture medium containing 10% serum was added. The cells were then cultured in a 37 ℃, 5% CO2 incubator.

[0054] Sixty h after transfection, the supernatant of 293T cells was collected. The supernatant was filtered through a 0.45 μm filter into an ultracentrifuge tube and centrifuged at 25,000 rpm for 2 h at 4 °C. The supernatant was then discarded, and the virus pellet was resuspended in viral preservation solution (purchased from Shanghai Jikai Gene). After thorough dissolution, the supernatant was collected after centrifugation at 10,000 rpm for 5 min at 4 °C. High-titer lentiviruses were obtained. The obtained lentiviruses were LV-SIRT5-RNAi (668632-1), LV-SSIRT5-RNAi (668633-1), and LV-SIRT5-RNAi (668634-1). The lentivirus titers are shown in Table 7.

[0055] Table 7 Lentiviral titers Meanwhile, sequence 5'TTCTCCGAACGTGTCACGT3' was constructed into the GV248 vector as a control group, and lentivirus was obtained by transfecting 293T cells according to the above method.

[0056] II. The effects of lentiviruses on cancer cells Cell information is shown in Table 8, and cell infection virus information is shown in Table 9.

[0057] Table 8 Cell Information Table 9 Information on Cell Infection with Viruses 1. Cell Culture Remove the TE10 cell cryovials from the liquid nitrogen tank and quickly place them in a 37°C water bath, shaking occasionally to thaw them as soon as possible. After complete thawing, disinfect the cryovials by wiping them with 75% alcohol and transfer them to a biosafety cabinet. Transfer the cryopreservation solution to a 15 ml centrifuge tube, add an equal volume of RPMI 1640 + 10% FBS medium, centrifuge at 1000 rpm for 5 min, remove the supernatant, and resuspend the cells in 1 ml of RPMI 1640 + 10% FBS medium. Seed the cell suspension into a culture flask containing 4 ml of complete medium, gently shake to mix, and place in a 37°C, 5% CO2 incubator. After 24 h, replace the RPMI 1640 + 10% FBS medium and continue culturing. Once the cell confluence reaches approximately 80%, passage the cells to maintain good cell growth.

[0058] 2. Lentiviral infection of cells Culture cells using standard methods to ensure good cell condition. Digest cells and count them the day before lentiviral infection, at a ratio of 1 × 10⁶ cells per well. 5 Cells were seeded into 6-well plates (antibiotic-free medium); according to Table 10, when the cell confluence was about 20%, the original medium was aspirated, and 1 ml of medium containing infection enhancement solution (purchased from Shanghai Jikai Gene) was added. At the same time, virus solution was added at MOI = 20 (see Table 11). Referring to the preliminary experimental results, the medium was replaced with medium containing 10% FBS after 16 h of lentiviral infection; after 72 h of infection, the infection efficiency was observed under an inverted fluorescence microscope; puromycin (final concentration 2 μg / ml) was added to screen for stable infected cell lines.

[0059] Table 10 Cell infection parameters Table 11 Virus Dosage Fluorescence imaging results (see) Figure 4 The image shows that the fluorescence information corresponds to the amount of virus used in Table 11.

[0060] 3. In cells SIRT5 Gene expression analysis 3.1 RT-qPCR detection SITR5 Gene expression Cells in the Sh-SIRT5#1, Sh-SIRT5#2, Sh-SIRT5#3, and Sh-NC groups were detected by RT-qPCR. SIRT5 Gene expression status. Internal reference genes. GAPDH Primers are shown in Table 1. SIRT5Gene primers are shown in Table 2, reaction system is shown in Table 4, and reaction conditions are as follows: 95 ℃ for 30 sec; 95 ℃ for 5 sec, 60 ℃ for 30 sec, 40 cycles; 95 ℃ for 15 sec, 60 ℃ for 1 h, 95 ℃ for 5 sec (for melting curve detection); using F = 2. -ΔΔCt Relative quantitative data analysis was performed (ΔCt = target gene Ct value - internal reference gene Ct value; -ΔCt = average ΔCt of the Sh-NC group - ΔCt value of each Sh-SIRT5 group; 2) -ΔΔCt This reflects the relative expression level of the target gene in each Sh-SIRT5 group compared to the Sh-NC group.

[0061] The above experimental results (see) Figure 5 A) indicates that, compared with the Sh-NC group cells, infection with Sh-SIRT5#2 and Sh-SIRT5#3 lentiviruses significantly inhibited cell cell growth. SIRT5 Gene expression.

[0062] 3.2 Western blot detection of SITR5 protein expression The expression of SIRT5 protein in cells of the Sh-SIRT5#1, Sh-SIRT5#2, Sh-SIRT5#3 and Sh-NC groups was detected by Western blot.

[0063] (1) Protein extraction: Pre-cooled benchtop high-speed low-temperature centrifuge; prepare protein lysis buffer (operate on ice): 1 ml RIPA lysis buffer + 10 μl protease inhibitor + 10 μl phosphatase inhibitor. Place cell culture flasks on ice, wash twice with PBS, aspirate excess liquid, add 100 μl protein lysis buffer to each culture flask, scrape off cells with a cell scraper, transfer to EP tubes, lyse on ice for 30 min, centrifuge at 4 ℃ and 14000 rpm for 15 min. Aspirate the supernatant and transfer to a new EP tube, add 5× loading buffer, boil for 5 min, centrifuge, and store in a -80 ℃ ultra-low temperature freezer.

[0064] (2) Protein quantification: The BCA method was used for quantification. The specific operation was performed according to the kit instructions. The standard protein (2 mg / ml) was serially diluted with PBS to set 7 protein concentration values ​​(0 μg / ml~2000 μg / ml). Solution A and solution B were prepared at a ratio of 1:50. The working solution volume was calculated according to (7 + number of samples) × 200 μl. 200 μl was added to each well of the 96-well plate. Then, 25 μl of serially diluted protein concentration solution was added to each well. 20 μl of PBS + 5 μl of protein sample was added to the remaining wells. The plate was incubated at 37 ℃ for 30 min. The 562 nm optical density value was measured with a microplate reader. A standard curve was prepared and the protein concentration was calculated. (3) Protein electrophoresis: Different concentrations of separating gels were prepared according to the molecular weight of the proteins. In this experiment, 8% separating gel and 5% stacking gel were used. The electrophoresis glass plate was installed, 8% separating gel was poured in, water was added to seal the gel, and it was left to stand for 30 min. The filter paper was used to absorb the liquid, the stacking gel was poured in, and the comb was immediately inserted. The plate was placed in the electrophoresis tank, and electrophoresis was performed at a constant voltage of 100 V for 90 min.

[0065] (4) Transfer: Soak the PVDF membrane in methanol for 10 min. Install the transfer clamp in the following order: sponge pad, filter paper, adhesive, PVDF membrane, filter paper, sponge pad. Remove air bubbles during installation. Place the transfer clamp in the transfer tank and transfer the membrane at a constant current of 300 mA for 90 min.

[0066] (5) Blocking, antibody incubation and chemiluminescence detection: Place the membrane in 5% skim milk powder and block it on a shaker at room temperature for 60 min; rinse the membrane with 1×TBST, then place the membrane in diluted primary antibody and incubate overnight on a shaker at 4 ℃; wash with TBST 3 times, 10 min each time; add secondary antibody and incubate on a shaker at room temperature for 1 h; wash with TBST 3 times, 10 min each time; prepare chemiluminescence solution 1:1 according to the instructions of Kangwei Century Chemiluminescence Detection Kit, drop it onto the membrane, and scan with a chemiluminescence imager.

[0067] The above experimental results (see) Figure 5 (B) indicates that, compared with the Sh-NC group cells, infection with Sh-SIRT5#2 and Sh-SIRT5#3 lentiviruses significantly inhibited the expression of SIRT5 protein in cells.

[0068] 4. Effects of Lentiviral Viruses on the Proliferation of Esophageal Squamous Cell Carcinoma Cancer Cells: TE10 cells in the logarithmic growth phase of the Sh-NC, Sh-SIRT5#2, and Sh-SIRT5#3 groups were digested with trypsin, and digestion was terminated with RPMI 1640 + 10% FBS medium. The cells were then resuspended and counted. 100 μl of each cell suspension was placed in a 96-well plate. Three replicates were made for the Sh-NC, Sh-SIRT5#2, and Sh-SIRT5#3 groups. According to the experimental design, five 96-well plates were prepared. After the cells had settled completely, the cell density of each experimental group was observed under a microscope. If the density was uneven, one group was fixed, and the cell amount of the other groups was adjusted and the plates were re-platened (e.g., if the Sh-NC group had more cells, the cell amount was reduced and the plates were re-platened). The plates were then placed in a cell culture incubator for culture. Starting from the day after plate formation, 10 μl of CCK-8 reagent was added to each well at 0 h, 12 h, 24 h, 36 h, and 48 h, followed by incubation at 37 ℃ for 2 h. OD values ​​were measured at 450 nm using a microplate reader. All data were analyzed using GraphPad Prism 8.3.0 software.

[0069] The above experimental results are as follows Figure 6The figure shows a comparison of the changes in light absorbance at 450 nm over time between the experimental groups (Sh-SIRT5#2 and Sh-SIRT5#3 groups) and the control group (Sh-NC group). The results show that, compared to the Sh-NC group, the proliferation of cells in the Sh-SIRT5#2 and Sh-SIRT5#3 groups was slowed, indicating that the lentiviruses in the Sh-SIRT5#2 and Sh-SIRT5#3 groups have an inhibitory effect on the proliferation of esophageal squamous cell carcinoma cells. SIRT5 Gene expression can significantly inhibit the proliferation of esophageal squamous cell carcinoma cells.

[0070] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0071] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. SIRT5 The application of gene inhibitors in the preparation of drugs for treating esophageal squamous cell carcinoma is characterized by, The SIRT5 Gene inhibitors SIRT5 The gene-interfering molecule is the only effective component; the interfering molecule contains a sense strand and an antisense strand; The sense strand of the interfering molecule is shown in SEQ ID NO.10, and the antisense strand is shown in SEQ ID NO.11; Alternatively, the sense strand of the interfering molecule is shown in SEQ ID NO.12, and the antisense strand is shown in SEQ ID NO.

13.

2. As described in claim 1 SIRT5 The application of gene inhibitors in the preparation of drugs for treating esophageal squamous cell carcinoma is characterized by, The drug is used to inhibit the proliferation of esophageal squamous cell carcinoma cells.

3. As described in claim 1 SIRT5 The application of gene inhibitors in the preparation of drugs for treating esophageal squamous cell carcinoma is characterized by, The SIRT5 The interference molecular target sequence of the gene is SIRT5-RNAi shown in SEQ ID NO.6 or SIRT5-RNAi shown in SEQ ID NO.

7.

4. The method according to claim 3 SIRT5 The application of gene inhibitors in the preparation of drugs for treating esophageal squamous cell carcinoma is characterized by, The interfering molecule targeting SIRT5-RNAi shown in SEQ ID NO. 6 contains the sense strand shown in SEQ ID NO. 10 and the antisense strand shown in SEQ ID NO. 11; The interfering molecule targeting SIRT5-RNAi shown in SEQ ID NO.7 contains the sense strand shown in SEQ ID NO.12 and the antisense strand shown in SEQ ID NO.

13.

5. An interfering molecule, characterized in that, The interfering molecule comprises a sense strand and an antisense strand, which are connected by a stem-loop structure to form a hairpin structure. The sense strand of the interfering molecule is shown in SEQ ID NO.10, and the antisense strand is shown in SEQ ID NO.11; Alternatively, the sense strand of the interfering molecule is shown in SEQ ID NO.12, and the antisense strand is shown in SEQ ID NO.

13.

6. A kind SIRT5 Gene inhibitors, characterized in that, The SIRT5 The gene inhibitor is a lentivirus containing the interfering molecule as described in claim 5.

7. The method according to claim 6 SIRT5 Gene inhibitors, characterized in that, The lentivirus is obtained by linking the interfering molecule with the GV248 vector, transforming competent cells, screening for interfering plasmids, transfecting 293T cells through a lentivirus three-plasmid packaging system, collecting the supernatant, and purifying and concentrating it.

8. An interfering molecule as described in claim 5 or any one of claims 6-7 SIRT5 Application of gene inhibitors in the preparation of inhibitors for esophageal squamous cell carcinoma.

Images