Application of LOC339524 gene and its encoded protein in the preparation of drugs for treating cancer

Abstract

The present invention relates to the technical field of cancer therapeutic drugs, and specifically to the use of the LOC339524 gene and its encoded protein in the preparation of drugs for treating cancer. This technical solution improves the transcription level of the LOC339524 gene and the expression level of the LOC339524 protein in human liver cancer cells, non-small cell lung cancer cells and glioma cells, thereby achieving the inhibitory effect on the above three cancer cells, which is mainly manifested in the significant inhibitory effect on cancer cell proliferation, migration and invasion. This technical solution uses LOC339524 as a target for the prevention and / or precise treatment of non-small cell lung cancer cells, liver cancer cells and gliomas for the first time, providing a new idea for the establishment of treatment plans for the above three cancers. This technical solution can solve the technical problem of the lack of new effective targets and related drugs for lung cancer, liver cancer and gliomas in the existing technology, and has an ideal prospect for promotion and application.

Description

Technical Field

[0001] The present invention relates to the technical field of cancer therapeutic drugs, and in particular to the use of the LOC339524 gene and its encoded protein in the preparation of drugs for treating cancer. Background Art

[0002] Lung cancer, liver cancer, and gliomas are highly malignant, with rich vascular tissues and a high propensity for metastasis and recurrence. Currently, surgical resection is the primary treatment for these tumors, supplemented by radiotherapy and chemotherapy. However, these tumors often invade and infiltrate early, blurring the boundary with surrounding normal tissue, making complete surgical resection difficult and leading to high postoperative recurrence rates. Advances in science and technology have led to the application of high-throughput technologies such as third-generation sequencing, single-cell transcriptomics, single-cell proteomics, and spatiotemporal multi-omics. Targeted therapies have emerged as a novel approach for treating malignant tumors, in addition to traditional treatments. These therapies disrupt specific target genes and / or proteins required for tumor development and progression, thereby inhibiting cancer cell growth and promoting cell death. Targeted therapies offer high specificity, significant efficacy, and minimal adverse reactions, but they require the identification of specific molecular markers as therapeutic targets. Although targeted drugs are currently available, liver cancer, lung cancer, and gliomas are highly heterogeneous, with multiple genes involved in their development and numerous potential therapeutic targets. Consequently, the development of detection reagents and drugs targeting new and effective targets is urgently needed.

[0003] The nucleotide sequence of LOC339524, obtained during the human genome sequencing in 2004, consists of 39,440 bases and is located on chromosome 1p22.3. Previously, the LOC339524 gene was thought to be a non-protein-coding RNA (LINC01140). However, the inventors, through CRISPR-directed genome knock-in, recombinant expression, and mass spectrometry analysis, have demonstrated for the first time that LOC339524 encodes a protein. The LOC339524 protein consists of 276 amino acids with a molecular weight of 28,694 Da. Using the monoclonal antibody 5-D3 (Patent No. ZL 2013 1 0078163.7) against human LOC339524, the expression, distribution, and related functions of the LOC339524 protein were preliminarily investigated using tissue microarrays. Results revealed that the LOC339524 protein is expressed in major human tissues and organs, including the heart, brain, lungs, liver, and kidneys, and its expression can be detected in human brain neurons. The role and mechanism of the LOC339524 gene and protein in the development and progression of diseases such as lung cancer, liver cancer, and glioma remain unclear. Further in-depth research is urgently needed to determine whether the LOC339524 gene and protein can serve as therapeutic targets for various cancers and whether they have the potential to develop drugs to treat related cancers or reagents to detect related diseases. Summary of the Invention

[0004] The purpose of the present invention is to provide the use of the LOC339524 gene and its encoded protein in the preparation of drugs for treating cancer, so as to solve the technical problem in the prior art of lacking new effective targets and related drugs for lung cancer, liver cancer and glioma.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] Application of the LOC339524 gene and its encoded protein in the preparation of drugs for treating cancer.

[0007] Furthermore, the nucleotide sequence of the LOC339524 gene is shown in SEQ ID NO.1, and the optimized nucleotide sequence of the LOC339524 gene is shown in SEQ ID NO.2.

[0008] Furthermore, the amino acid sequence of the LOC339524 protein is shown in SEQ ID NO.3 or SEQ ID NO.4.

[0009] Furthermore, the cancer is lung cancer, liver cancer or glioma.

[0010] Furthermore, the lung cancer is non-small cell lung cancer, the liver cancer is hepatocellular carcinoma, and the glioma is glioblastoma.

[0011] Furthermore, the amount of the LOC339524 gene or LOC339524 protein is increased in cancer cells.

[0012] Furthermore, the amount of the LOC339524 gene or LOC339524 protein in cancer cells is increased in an overexpressed manner.

[0013] Furthermore, the overexpression method is: the cancer cells are transfected with an expression vector, wherein the expression vector is integrated with the LOC339524 gene.

[0014] Furthermore, an expression vector for expressing the protein shown in SEQ ID NO. 3 or SEQ ID NO. 4 is constructed; and then the expression vector is transfected into cancer cells.

[0015] Furthermore, the drug is used to inhibit cancer cell proliferation, migration and invasion.

[0016] The technical principle and beneficial effects of this technical solution are:

[0017] This technical solution optimizes the LOC339524 gene sequence, then constructs an expression vector for the gene and transfects it into cancer cells. Overexpression of the LOC339524 protein in cancer cells can produce a significant inhibitory effect on three types of cancer cells: lung cancer, liver cancer, and glioma, mainly manifested in the inhibition of cell proliferation, migration, and invasion. It can be seen that increasing the transcription level of the LOC339524 gene and the expression level of the LOC339524 protein in cancer cells can be used to treat diseases such as non-small cell lung cancer, hepatocellular carcinoma, and glioblastoma. The LOC339524 gene or protein can also be used as a therapeutic target for the treatment of corresponding cancers, and can be used for the screening and design of related drugs, providing new ideas for the treatment of non-small cell lung cancer cells, liver cancer cells, and gliomas, and has good application prospects.

[0018] Experiments in this technical solution have demonstrated that the LOC339524 protein has the effect of inhibiting the proliferation, metastasis, and invasion of cancer cells, and can therefore be used as a drug for the treatment of cancer. It should be emphasized that the LOC339524 gene can express non-coding RNA and can also encode proteins, but the long-chain non-coding RNA (lncRNA) produced by the LOC339524 gene and the LOC339524 protein are two completely different substances. Long-chain non-coding RNA (lncRNA) is a type of non-coding RNA with a length greater than 200 nucleotides, and its essence is nucleotides. The essence of LOC339524 protein is amino acids. The non-coding RNA and protein encoded by the same gene are two substances with completely different material bases, and their effects and mechanisms of action are very different.

[0019] It has been reported that the non-coding RNA LINC01140 (lncRNA01140) encoded by the LOC339524 gene promotes lung cancer progression and tumor immune escape by directly acting on miRNA (reference: Rongmu Xia, LINC01140 promotes the progression and tumor immune escape in lung cancer bysponging multiple microRNAs, J Immunother Cancer. 2021Aug 25; 9(8):e002746.doi:10.1136 / jitc-2021-002746). The LOC339524 protein in this protocol is a new protein encoded by lncRNA01140, and its role in cancer has not been reported in the literature. The experimental results showed that overexpression of LOC339524 protein has the effect of inhibiting cancer cells, which is contrary to the functional phenotype of lncRNA01140 in promoting lung cancer progression. Although the tumor suppressor mechanism of LOC339524 protein has not yet been deeply explored, based on the antagonistic function of LOC339524 protein and lncRNA01140 in cancer cells (the trend of action is opposite), we speculate that LOC339524 protein may exert its tumor suppressor effect through a molecular mechanism independent of lncRNA01140, and therefore can be used as a drug for cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the structure of the expression vector pCAGGS-0580-Flag in Example 2.

[0021] Figure 2 These are the experimental results of WB detection of transfected cells in Example 3 (pCAGGS: empty vector; pCAGGS-LOC-Flag: vector integrated with SEQ ID NO. 1; pCAGGS-0580-Flag: vector integrated with SEQ ID NO. 2).

[0022] Figure 3 These are the research results of Example 4 on the effect of overexpression of LOC339524 protein on cell proliferation in three cancer cells (pCAGGS: empty vector; pCAGGS-LOC-Flag: vector integrated with SEQ ID NO. 1; pCAGGS-0580-Flag: vector integrated with SEQ ID NO. 2).

[0023] Figure 4These are the research results of Example 4 on the effect of overexpression of LOC339524 protein on cell migration ability in three cancer cells (pCAGGS: empty vector; pCAGGS-LOC-Flag: vector integrated with SEQ ID NO. 1; pCAGGS-0580-Flag: vector integrated with SEQ ID NO. 2).

[0024] Figure 5 These are the research results of Example 4 on the effect of overexpression of LOC339524 protein on cell invasion ability in three cancer cells (pCAGGS: empty vector; pCAGGS-LOC-Flag: vector integrated with SEQ ID NO. 1; pCAGGS-0580-Flag: vector integrated with SEQ ID NO. 2). DETAILED DESCRIPTION

[0025] The present invention will be further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used are all commercially available. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art.

[0026] Example 1: Discovery and optimization of the LOC339524 coding region sequence

[0027] The nucleotide sequence of the coding region of the LOC339524 gene is SEQ ID NO.1, and the optimized nucleotide sequence of the coding region is SEQ ID NO.2.

[0028] SEQ ID NO.1:

[0029] ATGCTGGCCCGCCGGGACCTGGGACTTGTGCCACATGGAGTGTCGGGAGTCTCCATTGCCGCGAGTTCTACACCACAGGGCCAGGCTGTTTGCTCCCCATCGGTCGCTGCCCCCAGCACCCTGTTGTTATTAAGGACTCATTTGCTTGGAGCGGCATCATTACAAGGGTGTGGGGTACTACATATACTCCCTATTTTTCTATTTTCGAAAGGCTGCAGGCGCGATGCACAGTGCGCTTGCACGGTGGGGCCTAGTGCTAGCCCGAGGAGCGGACGGGGGCCGGGCAGGGGCGGTGGGCGCCGGCCTCGTCTCGGTGCTGCTCGGTCAGGCTGTCCCGGCGCGGCGGCCGCGGGAGGCCCTGCCGTCCTTCACCCCTGGAGGCGGGCTGGGGGGCGGGTGCGGGGCGCTTCCCCTCCTCAGGGCCCTCAAACCGCAAGGGGGTTTCCGCTTCCCAGTCGATGGTCGTCCTCTCCCATCCCCGGCTGCATCTCCATTTACCCGTCGCCCATTTCCTTTGCCCATCCAGGCTCCTTGGCTCCACTGGGGTCTCCGTTCCCTTCTCCCGGTCCCCCCTCCAGGTCGCGGCTCCTTTGTCCAGGACTACGCAGGGGCTTGACCCCAGGGCGCTGGTTTAGGCCGGATCTGGGGTCCCTTGTCACTCCCAGGCTTCTTCCACTTCCGAATTCTGGAGAACCGGGAATCAAGCCCTGCGCGTTCCTCTTCTTCCTCCTTCGTGCCGAAAGCACGCTTCATGTCTGCCAGGGCATCAGTTCTGAAAGTGAGCGGAGAACAAGGAGTTTCTTTTTCTTCCCCAGAAGTTGCCTTTTGTAA;

[0030] SEQ ID NO.2:

[0031] ATGCTGGCCAGGAGGGACCTGGGCCTGGTGCCTCACGGCGTGTCCGGAGTGTCCATCGCCGCCTCCAGCACACCCCAGGGCCAGGCTGTGTGTAGCCCTAGCGTGGCCGCCCCTAGCACCCTGCTGCTGCTGAGGACACACCTGCTGGGCGCCGCCAGCCTGCAGGGATGTGGAGTGCTGCACATCCTGCCTATCTTCCTGTTTTCCAAGGGCTGTAGGAGAGATGCCCAGTGCGCCTGTACAGTGGGCCCTTCCGCCAGCCCCAGAAGCGGAAGAGGCCCCGGAAGAGGCGGCGGAAGAAGGCCAAGGCTGGGCGCTGCCAGGTCCGGATGTCCCGGAGCTGCCGCTGCGGGCGGACCAGCTGTGCTGCACCCCTGGAGGAGAGCCGGCGGAAGGGTGAGGGGCGCTAGCCCTCCACAGGGCCCACAGACCGCCAGAGGCTTCCCTCTGCCCTCCAGATGGAGCAGCTCCCCCATCCCTGGCTGTATCTCCATCTACCCCTCCCCTATCTCCTTTGCCCACCCCGGCTCCCTGGCCCCACTGGGATCTCCATTCCCTTCCCCTGGCCCCCCTTCCAGATCCAGACTGCTGTGTCCTGGCCTGAGGAGGGGCCTGACCCCCGGAAGATGGTTTAGACCTGACCTGGGCTCCCTGGTGACACCCAGGCTGCTGCCTCTGCCCAACTCCGGCGAGCCCGGAATCAAGCCCTGTGCCTTCCTGTTTTTTCTGCTGAGAGCCGAGAGCACCCTGCACGTGTGCCAGGGCATCTCCAGCGAGTCCGAGAGAAGGACAAGGAGCTTCTTCTTCTTTCCTAGGAGCTGCCTGCTGTAA。

[0032] Example 2: Construction of Expression Vector

[0033] The base sequence shown in SEQ ID NO. 2 was chemically synthesized by Wuhan Jinkairui Bioengineering Co., Ltd., and restriction sites were introduced at both ends. The empty vector pCAGGS (Beijing Solaibao, AT000109-1EA) was integrated with the synthesized gene fragment to form an expression vector. The specific process is as follows:

[0034] (1) Enzyme digestion of pCAGGS and target gene: 1 μg of pCAGGS or target gene was added to 5 μl 10× cutsmart buffer (BioLab, SV0827), 1 μl BstXI enzyme (Thermo Scientific TM , ER1021), 1 μl BglII enzyme (Thermo Scientific TM , ER0081), add double distilled water to 50 μl, and digest at 37°C for 2 h. The digestion products were detected by electrophoresis and recovered according to the GeneJET Gel Recovery Kit (Thermo Scientific) TM , K0691).

[0035] (2) Ligation of pCAGGS and target gene: 50 ng of pCAGGS after enzyme digestion and 1 μg of target gene after enzyme digestion were added to 2 μl of 10×T4 DNA Ligase buffer (Thermo Scientific TM , B69), 1 μl T4 DNA Ligase (Thermo Scientific TM , EL0014), add double-distilled water to make up to 20 μl, and connect at 16°C for 1 h.

[0036] (3) Conversion:

[0037] The ligation product to be transformed was added to a culture medium containing TOP10 competent cells (Thermo Scientific TM, C404006) (50μl competent cells require 25ng DNA), the volume should not exceed 5% of the competent cells, gently rotate several times to mix the contents, and ice bath for 30min. Place the centrifuge tube mixture in circulating water heated to 42°C, heat shock for 90s, do not shake the tube. Quickly transfer the tube to an ice bath and allow the cells to cool for 1min. Add 200μl SOC liquid culture medium to each tube, warm the culture medium to 37°C with a water bath, then transfer the tube to a shaker set at 37°C and culture at 220rpm for 45min to allow the cells to recover and express the resistance marker gene encoded by the plasmid. Transfer an appropriate volume (200μl per 90mm plate) of transformed competent cells to LB culture medium containing the corresponding antibiotics. Invert the plate and culture at 37°C. Plaques will appear after 16 hours.

[0038] (4) Colony PCR verification: After colonies grow on the plate, randomly pick several colonies and perform colony PCR verification to detect transformants.

[0039] (5) Sequencing verification: The positive clones were sent to Wuhan Jinkairui Bioengineering Co., Ltd. for sequencing verification. The clones with the correct verification were retained, which contained the pCAGGS-0580-Flag expression vector ( Figure 1 ; A: expression vector map; B: expression vector sequence). The expression vector expresses the LOC339524 protein, and a FLAG tag is added to the C-terminus of the protein. The specific sequence of the LOC339524 protein is shown in SEQ ID NO.3:

[0040] MLARRDLGLVPHGVSGVSIAASSTPQGQAVCSPSVAAPSTLLLLRTHLLGAASLQGCGVLHILPIFLFSKGCRRDAQCACTVGPSASPRSGRGPGRGGGRRPRLGAARSGCPGAAAAGGPAVLHPWRRAGGRVRGASP PQGPQTARGFPLPSRWSSSPIPGCISIYPSPISFAHPGSLAPLGSPFPSPGPPSRSRLLCPGLRRGLTPGRWFRPDLGSLVTPRLLPLPNSGEPGIKPCAFLFFLLRAESTLHVCQGISSESERRTRSFFFFPRSCLL.

[0041] The overall protein sequence after adding the FLAG tag is shown in SEQ ID NO.4 (the underlined part is the FLAG tag):

[0042] MLARRDLGLVPHGVSGVSIAASSTPQGQAVCSPSVAAPSTLLLLRTHLLGAASLQGCGVLHILPIFLFSKGCRRDAQCACTVGPSASPRSGRGPGRGGGRRPRLGAARSGCPGAAAAGGPAVLHPWRRAGGRVRGASP PQGPQTARGFPLPSRWSSSPIPGCISIYPSPISFAHPGSLAPLGSPFPSPGPPSRSRLLCPGLRRGLTPGRWFRPDLGSLVTPRLLPLPNSGEPGIKPCAFLFFLLRAESTLHVCQGISSESERRTRSFFFFPRSCLL DYKDDDDK .

[0043] Example 3: Expression vector transfection

[0044] A549 (human non-small cell lung cancer cell line), HepG2 (hepatocellular carcinoma cell line), and U87mg (glioblastoma cell line) cells were adjusted to 2.5 × 10 cells / mL using complete culture medium (Hyclone, SH30022.01) containing 10% fetal bovine serum (Lonsera, S711-0011S). 5 1 μg / ml was inoculated into a 6-well plate, 2 ml per well. After 24 hours of culture, the cell confluence was 70-80%. 3 μL PolyJetTM (SignaGen, SL100489) and 1 μg pCAGGS-0580-Flag or pCAGGS were diluted in 200 μl serum-free medium, mixed gently, and allowed to stand at room temperature for 5 minutes. The serum-free medium containing PolyJetTM was added to the serum-free medium containing pCAGGS-0580-Flag or pCAGGS, and allowed to stand at room temperature for 15 minutes (no more than 30 minutes) to obtain the transfection mixture. During the waiting process of the above steps, the medium in the 6-well plate was aspirated and gently washed with 2 ml 1×PBS. After aspiration, 1.6 ml of fresh complete medium was added. After standing at room temperature, the transfection mixture was added to the 6-well plate and placed in a cell culture incubator. 24 h after transfection, pCAGGS-0580-Flag-transfected cells and pCAGGS-transfected cells were obtained and used for subsequent experiments.

[0045] Western Blot analysis was performed on the cells transfected 24 hours after transfection (pCAGGS-0580-Flag transfected cells and pCAGGS transfected cells) to assess the expression level of LOC3339524 protein. The specific steps are as follows:

[0046] (1) Total protein extraction and quantification

[0047] Aspirate the culture medium from the 6-well plate and gently wash with 2 ml of 1x ice-cold PBS. Repeat this process once. After aspirating the PBS, add 100 μl of RIPA lysis buffer (containing 1 μl of PMSF) and lyse the cells on ice for 30 min. Transfer the cell lysate to a 1.5 ml EP tube and centrifuge at 12,000 rpm for 15 min at 4°C. Gently aspirate the supernatant, which is the total cellular protein, and store at -80°C.

[0048] (2) Protein quantification

[0049] Total cell protein was quantified using the BCA protein concentration assay kit (Biyuntian, P0009). Add 1.2 ml of protein standard solution to a tube of protein standard (30 mg BSA) and dissolve thoroughly to prepare a 25 mg / ml protein standard solution. Dilute an appropriate amount of the 25 mg / ml protein standard to a final concentration of 0.5 mg / ml. For example, add 20 μl of the 25 mg / ml protein standard to 980 μl of the diluent to prepare a 0.5 mg / ml protein standard. Prepare the BCA working solution based on the sample quantity, using a ratio of 50:1 (BCA Reagent A: Reagent B) and mix thoroughly. Note: The BCA working solution is stable at room temperature for 24 hours. Add 0, 1, 2, 4, 8, 12, 16, and 20 μl of the standard to the standard wells of a 96-well plate. Add standard diluent to a total of 20 μl, corresponding to standard concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / ml, respectively. Add an appropriate volume of sample to the sample wells of the 96-well plate. If the sample volume is less than 20 μl, add standard diluent to a total of 20 μl. Add 200 μl of BCA working solution to each well and incubate at 37°C for 30 minutes. Measure the absorbance at the A562 wavelength using a microplate reader. Calculate the cell protein concentration based on the standard curve and the sample volume used, and quantify to the nearest 5 μg / μl. Add 5× SDS-PAGE protein loading buffer (Biyuntian, P0285) to the cell protein at a ratio of 1:4 by volume. Mix thoroughly, boil in boiling water for 15 minutes, and store at -20°C.

[0050] (3) Western Blot

[0051] Gel preparation: Secure two clean glass plates to the gel preparation tank. Add ddH2O and observe for leakage. If no leakage is observed, remove any ddH2O from the inner side of the glass plates with filter paper and prepare for gel casting. Based on the molecular weight of the protein, prepare the appropriate volumes of 10% separating gel (7 mL / plate) and 5% stacking gel (2 mL / plate) according to the instructions in the gel preparation kit (Biyuntian, P0012AC). After the stacking gel is poured, immediately insert the sample comb, taking care to avoid air bubbles. Let stand at room temperature for 20 minutes before loading the sample.

[0052] Add samples: Pour freshly prepared 1× electrophoresis buffer into the sample wells until it covers the sample wells. Use a micropipette to load 20 μg of each sample.

[0053] Electrophoresis: Connect the positive and negative electrodes, 80V for stacking gel and 120V for separation gel, constant voltage electrophoresis. TM , 26616), stop electrophoresis when the target band reaches the middle of the separation gel and prepare for membrane transfer.

[0054] Transfer: Cut the entire block of separation gel and cut the PVDF membrane to size. Activate the PVDF membrane (Millipore, GVWP04700) with methanol for 20 seconds before assembling the "sandwich." Assemble the "sandwich" in the following order: thick filter paper, PVDF membrane, separation gel, filter paper, and sponge (no air bubbles between the PVDF membrane and separation gel). Place the membrane in the transfer apparatus according to color and transfer at 200 mA constant current for 150 minutes.

[0055] Blocking: Immerse the transferred PVDF membrane in 5% skim milk and block at room temperature for 120 minutes.

[0056] Incubation with primary antibodies: Wash the PVDF membrane once with TBST. Cut the target band according to the pre-stained protein marker and place it in an antibody incubation box containing diluted Flag primary antibodies (CST, 14793S) and β-actin (Wuhan Tri-Tech, 66009-1-Ig) at 4°C overnight.

[0057] Incubation with secondary antibody: Remove the strips incubated with primary antibody yesterday and wash them four times with TBST on a decolorizing shaker for 5 minutes each. After rinsing, place the strips in an antibody incubation box containing diluted secondary antibody (Wuhan Tri-Tek, SA00001-4) at room temperature for 2 hours.

[0058] Development: Solution A and Solution B from the ECL kit (Chongqing Baoguang) were mixed in a 1:1 ratio in a brown glass bottle. The strips were placed on the lid of a well plate covered with sealing film. The developer solution was added dropwise, and the image was acquired after development and exposure. Grayscale values ​​of the strips were analyzed using ImageJ software.

[0059] The results of LOC339524 protein expression are shown in Figure 2 (A: WB image; B: ImageJ quantitative grayscale value). The results showed that compared with cells transfected with pCAGGS (marked as pCAGGS in the figure), the expression level of LOC339524 protein in cells transfected with pCAGGS-0580-Flag (marked as pCAGGS-0580-Flag in the figure) was significantly increased, and the difference was statistically significant (P<0.01).

[0060] Example 4: Cell Phenotype after High Expression

[0061] (1) Proliferation assay

[0062] After trypsin digestion, the cells were collected by centrifugation at 800 rpm for 5 minutes and inoculated into a 96-well plate at 1500 cells / well, 100 μl / well. After 24 hours, the cell confluence was about 20% and transfection of pCAGGS-0580-Flag or pCAGGS (0.1 μg / well) was started. The transfection method is shown in Example 3. The general process is as follows: the transfection mixture is prepared according to the proportion of Example 3, and 100 μl of the transfection mixture is added to each well of the 96-well plate. The cell growth state after transfection of pCAGGS-0580-Flag or pCAGGS was observed under an inverted optical microscope. The cells were placed under an inverted optical microscope for observation and photographed. CCK8 evaluated the number of cells obtained 24 hours, 48 ​​hours and 72 hours after transfection.

[0063] Prepare 100 μl of CCK8 solution (Biyuntian, C0041) per well, using a volume ratio of CCK8 to serum-free medium of 1:9. Aspirate the medium, gently wash with 100 μl of 1× PBS, then aspirate and add the prepared CCK8 solution. Incubate at 37°C for 1 hour. Measure absorbance at 450 nm using a microplate reader to assess cell number.

[0064] The results of proliferation experiments were as follows Figure 3 Figures AC are photographs, and Figures DF are statistical graphs of CCK8 assay results. The results show that compared with cells transfected with pCAGGS (labeled as pCAGGS in the figure), cells transfected with pCAGGS-0580-Flag (labeled as pCAGGS-0580-Flag in the figure) showed a significant decrease in proliferation at 48 hours. The difference was statistically significant (P < 0.01), indicating that pCAGGS-0580-Flag transfection inhibited cell proliferation. This suggests that LOC339524 protein is overexpressed in cancer cells and has the effect of inhibiting cancer cell proliferation.

[0065] (2) Scratch test

[0066] The cells obtained 24 hours after transfection (pCAGGS-0580-Flag transfected cells and pCAGGS transfected cells) were subjected to the experiment. When the cell confluence reached 99-100%, a 200μl pipette tip was used to make a "cross" stroke perpendicular to the bottom of the well plate, with uniform force, trying to ensure uniform width and horizontal and vertical strokes. The cells were washed three times with PBS, serum-free medium was added, and the cells were photographed under an inverted optical microscope at 0h, 24h, 48h, and 72h. The migration rate and migration area ratio were calculated as follows:

[0067] Migration rate = (initial scratch area - 24h / 48 / 72h scratch area) / initial scratch area;

[0068] Migration area ratio = migration rate of the experimental group / migration rate of the control group.

[0069] The experimental group consisted of cells transfected with pCAGGS-0580-Flag, and the control group consisted of cells transfected with pCAGGS.

[0070] The migration experiment results are as follows Figure 4 Figures AC are photographs, and Figures DF are statistical graphs of the migration area ratio. The results show that compared with cells transfected with pCAGGS (labeled as pCAGGS in the figure), cells transfected with pCAGGS-0580-Flag (labeled as pCAGGS-0580-Flag in the figure) showed a significant decrease in migration rate at 24 and 48 hours. The difference was statistically significant (P < 0.01), indicating that pCAGGS-0580-Flag inhibited cell migration. This suggests that LOC339524 protein is overexpressed in cancer cells and has the effect of inhibiting cancer cell migration.

[0071] (3) Invasion assay

[0072] The cells obtained 24 hours after transfection (cells transfected with pCAGGS-0580-Flag and cells transfected with pCAGGS) were used for the experiment. At 4°C (on ice), Matrigel (BD, 356237) was diluted 1:8 with serum-free medium, and 60 μl was evenly added to the upper chamber surface of the bottom membrane of the Transwell chamber (Corning, 3422). It was incubated in a 37°C incubator for 3 hours. After the incubation was completed, the excess liquid in the upper chamber was aspirated and discarded. 100 μl of serum-free medium was added to each well and placed in the incubator for 30 minutes. After trypsin digestion, the cells were collected by centrifugation at 800 rpm for 5 minutes, the supernatant was discarded, and the cells were gently washed twice with PBS. Then, the cells were suspended with serum-free medium and the cell density was adjusted to 2.5×10 5 / ml. Add 500μL of complete culture medium to the lower chamber of the 24-well plate, then use tweezers to place the Transwell chamber in the 24-well plate, take 200μL of cell suspension and add it to the upper chamber, and finally place it in the incubator for culture for 24 hours. Use tweezers to remove the chamber, suck out the culture medium in the upper chamber, and gently wipe the Matrigel and the cells in the upper chamber with a cotton swab. Take a new 24-well plate and add 500μL of 4% paraformaldehyde (Biyuntian, P0099), put the chamber in and fix it at room temperature for 30 minutes. Remove the chamber, suck out the fixative in the upper chamber, move it to the well where 800ul of 1% crystal violet (Solabo, G1062) staining solution has been added in advance, and stain for 30 minutes. After staining, gently wipe the upper side of the chamber with a cotton swab to wipe off the dye that is non-specifically bound to the upper surface of the chamber.

[0073] 8) Gently wash and soak the chamber several times with PBS, remove the chamber, aspirate the liquid in the upper chamber, carefully wipe off the cells on the bottom membrane surface of the upper chamber with a wet cotton swab, and observe and photograph under an inverted microscope.

[0074] The invasion assay results were as follows Figure 5 As shown, Figure A is a photograph, and Figure B is a statistical chart of invasive cells. The results show that the number of cells transfected with pCAGGS (labeled as pCAGGS in the figure) and cells transfected with pCAGGS-0580-Flag (labeled as pCAGGS-0580-Flag in the figure) that passed through the Matrigel and basement membrane at 24 hours was significantly reduced, and the difference was statistically significant (P<0.01), indicating that pCAGGS-0580-Flag transfection can inhibit cell invasion. This indicates that LOC339524 protein is overexpressed in cancer cells and has the effect of inhibiting cancer cell invasion.

[0075] Comparative Example 1

[0076] Before codon optimization of the LOC339524 gene (SEQ ID NO. 1), the inventors also attempted to express SEQ ID NO. 1 in cells. The LOC339524 gene is human, and the cells overexpressing it in this protocol are also human. Theoretically, the gene of SEQ ID NO. 1 should also be well expressed in human cells. Generally speaking, codon optimization is performed based on codon bias. Codon optimization is a process that improves gene expression and enhances the translation efficiency of the target gene by adapting to the codon bias of the host organism. Codon bias specifically refers to the fact that different organisms use degenerate codons at different frequencies. Cells have fewer tRNAs corresponding to rare codons, and genes that frequently use these codons are easily hindered in translation, significantly reducing protein expression levels. If the gene's synonymous codon usage frequency matches that of the expression host, protein expression levels will be significantly improved. However, this protocol does not address the issue of codon bias between species. It involves expressing a human gene in human cells, so there are no existing guidelines for codon optimization.

[0077] However, after actually expressing the gene of SEQ ID NO.1 in human cells, it was found that the expression level of the gene of SEQ ID NO.1 in human cells was very low. High expression of the original sequence of the LOC339524 gene (SEQ ID NO.1) can also inhibit tumor proliferation, migration and invasion, but compared with the optimized sequence (SEQ ID NO.2), the technical effect is poor. The inventors analyzed that the reason is that when the same amount of plasmid is transfected, the optimized sequence can express more LOC339524 protein. For detailed experimental results, see Figure 2-Figure 5pCAGGS-LOC-Flag. The preparation method of pCAGGS-LOC-Flag is the same as that of pCAGGS-0580-Flag in Example 3, and the cell transfection is carried out in the same way, except that the gene sequence is replaced with SEQ ID NO.1. It can be seen from the experimental results that the expression level of the expression vector containing SEQ ID NO.1 in the cell is low, and its effect of inhibiting tumor proliferation, migration and invasion is poor. Although this scheme is to express human genes in human cells, converting the sequence of SEQ ID NO.1 into the sequence of SEQID NO.2 to express the same protein can greatly increase the protein expression level in human cells and achieve better cancer cell inhibition effect. This is something that the inventors did not expect. The use of the SEQ ID NO.2 sequence has achieved unexpected technical effects.

[0078] The above is only an embodiment of the present invention, and the common knowledge such as the specific structure and characteristics of the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several variations and improvements can be made without departing from the structure of the present invention, and these should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. Use of the LOC339524 gene and its encoded protein in the preparation of a drug for treating non-small cell lung cancer, hepatocellular carcinoma or glioblastoma, characterized in that: The nucleotide sequence of the LOC339524 gene is shown in SEQ ID NO.1, and the optimized nucleotide sequence of the LOC339524 gene is shown in SEQ ID NO.2; the amino acid sequence of the LOC339524 protein is shown in SEQ ID NO.3 or SEQ ID NO.

4.

2. Use of the LOC339524 gene and its encoded protein according to claim 1 in the preparation of a medicament for treating non-small cell lung cancer, hepatocellular carcinoma or glioblastoma, characterized in that: The LOC339524 gene or LOC339524 protein is overexpressed and increased in cancer cells.

3. Use of the LOC339524 gene and its encoded protein according to claim 2 in the preparation of a medicament for treating non-small cell lung cancer, hepatocellular carcinoma or glioblastoma, characterized in that: The overexpression method is: the cancer cells are transfected with an expression vector, wherein the expression vector is integrated with the LOC339524 gene.

4. Use of the LOC339524 gene and its encoded protein according to claim 3 in the preparation of a medicament for treating non-small cell lung cancer, hepatocellular carcinoma or glioblastoma, characterized in that: An expression vector for expressing the protein shown in SEQ ID NO. 3 or SEQ ID NO. 4 is constructed; and the expression vector is then transfected into cancer cells.

5. Use of the LOC339524 gene and its encoded protein according to any one of claims 1 to 4 in the preparation of a medicament for treating non-small cell lung cancer, hepatocellular carcinoma or glioblastoma, characterized in that: The drug is used to inhibit cancer cell proliferation, migration and invasion.

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