LOC339524 gene and application of encoding protein thereof in preparation of medicine for treating cancer

Abstract

The invention relates to the technical field of medicines for treating cancers, in particular to an application of a LOC339524 gene and an encoding protein thereof in preparing medicines for treating cancers. According to the technical scheme, the transcription level of LOC339524 genes in human liver cancer cells, non-small cell lung cancer cells and glioma cells and the expression quantity of LOC339524 protein are improved, and then the inhibition effect on the three cancer cells is achieved, and the inhibition effect is mainly shown in the remarkable inhibition effect on cancer cell proliferation, migration and invasion. According to the technical scheme, LOC339524 serves as a target spot for preventing and / or precisely treating non-small cell lung cancer cells, liver cancer cells and glioma for the first time, and a new thought is provided for establishment of treatment schemes of the three cancers. The technical scheme can solve the technical problem of lack of new effective targets and related drugs for lung cancer, liver cancer and glioma in the prior art, and has ideal popularization and application prospects.

Description

Technical Field

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

[0002] Lung cancer, liver cancer and glioma are highly malignant, with rich blood vessels in the tumor tissue, and are extremely prone to metastasis and recurrence. At present, the treatment for the above tumors often adopts the treatment method mainly based on surgical resection and supplemented by radiotherapy and chemotherapy. However, invasion and infiltration often occur at an early stage, the boundary with the surrounding normal tissues is blurred, it is difficult to completely remove surgically, and the postoperative recurrence rate is relatively high. With the development of technology, the application of high-throughput technologies such as third-generation sequencing, single-cell transcriptomics, single-cell proteomics and spatial-temporal multi-omics has emerged, and targeted therapy has become a new method for treating malignant tumors in addition to traditional treatment means. It prevents the growth of cancer cells and promotes death by interfering with specific target genes and / or proteins required for tumor development. Targeted therapy has strong specificity, obvious curative effect and few adverse reactions, but the above methods need to find specific molecular markers as treatment targets. Although there are currently targeted drugs on the market, liver cancer, lung cancer and glioma have high heterogeneity, their development involves multiple genes, and there are many potential treatment targets. Therefore, there is an urgent need to develop detection reagents and drugs for new effective targets.

[0003] The nucleotide sequence of LOC339524 was obtained during the human genome sequencing in 2004, consisting of 39,440 bases, and is located on chromosome 1p22.3. In the past, the LOC339524 gene was considered to be a non-protein-coding RNA (LINC01140), but the inventors first confirmed through methods such as CRISPR genome site-directed knock-in, recombinant expression, and mass spectrometry identification that LOC339524 can encode protein expression. The LOC339524 protein consists of 276 amino acids and has a relative molecular weight of 28,694 Da. Through the monoclonal antibody 5-D3 of human LOC339524 protein (patent number: ZL 2013 1 0078163.7), the expression, distribution and related functions of LOC339524 protein were preliminarily studied using tissue chips. The results showed that the LOC339524 protein was expressed in the main tissues and organs of the human body such as the heart, brain, lung, liver and kidney, and the expression of LOC339524 could be detected in human brain nerve cells. The roles and mechanisms of the LOC339524 gene and protein in the occurrence and development of diseases such as lung cancer, liver cancer and glioma are still unclear. Whether the LOC339524 gene and protein can be used as treatment targets for various cancers, whether they have the potential to develop drugs for treating related cancers or reagents for detecting related diseases, requires further in-depth research. Summary of the Invention

[0004] The object of the present invention is to provide the application of the LOC339524 gene and its encoded protein in the preparation of drugs for treating cancer, so as to solve the technical problems 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] The application of the LOC339524 gene and its encoded protein in the preparation of drugs for treating cancer.

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

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

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

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

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

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

[0013] Further, the overexpressed manner is that the cancer cells are transfected with an expression vector, and the expression vector integrates the LOC339524 gene.

[0014] Further, construct an expression vector for expressing the protein as shown in SEQ ID NO.3 or SEQ ID NO.4; then transfect the expression vector into cancer cells.

[0015] Further, the drug is used to inhibit the proliferation, migration and invasion of cancer cells.

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

[0017] This technical solution optimizes the LOC339524 gene sequence, then constructs an expression vector of this gene and transfects it into cancer cells. Overexpressing the LOC339524 protein in cancer cells can significantly inhibit three types of cancer cells, namely lung cancer cells, liver cancer cells and glioma cells, mainly manifested in the inhibition of cell proliferation, migration and invasion. Thus, increasing the transcriptional 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 multiforme. The LOC339524 gene or protein can also be used as a therapeutic target for treating the corresponding cancers, 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 having good application prospects.

[0018] Experimental verification of this technical solution shows that the LOC339524 protein has the function of inhibiting the proliferation, metastasis and invasion of cancer cells, and thus can be used as a drug for treating cancer. It should be emphasized that the LOC339524 gene can express non-coding RNA or encode proteins, but the long non-coding RNA (lncRNA) produced by the LOC339524 gene and the LOC339524 protein are two completely different substances. Long non-coding RNA (lncRNA) is a type of non-coding RNA with a length greater than 200 nucleotides, and its essence is nucleotides. While the essence of the 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 there are very large differences in their action effects and mechanisms.

[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 miRNAs (Reference: Rongmu Xia, LINC01140 promotes the progression and tumor immune escape in lung cancer by sponging multiple microRNAs, J Immunother Cancer. 2021 Aug 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 show that the overexpression of the LOC339524 protein has an inhibitory effect on cancer cells, which is opposite to the functional phenotype of lncRNA01140 promoting lung cancer progression. Although the anti-cancer mechanism of the LOC339524 protein has not been deeply explored at present, based on the antagonism of the functions of the LOC339524 protein and lncRNA01140 in cancer cells (the action trends are opposite), we speculate that the LOC339524 protein may play its anti-cancer role through a molecular mechanism independent of lncRNA01140. Therefore, it can be used as a drug for treating cancer. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0021] Figure 2 Experimental results of WB detection of transfected cells in Example 3 (pCAGGS: empty vector; pCAGGS-LOC-Flag: vector integrating SEQ ID NO.1; pCAGGS-0580-Flag: vector integrating SEQ ID NO.2).

[0022] Figure 3 Research results on the effect of overexpressing the LOC339524 protein on cell proliferation ability in three types of cancer cells in Example 4 (pCAGGS: empty vector; pCAGGS-LOC-Flag: vector integrating SEQ ID NO.1; pCAGGS-0580-Flag: vector integrating SEQ ID NO.2).

[0023] Figure 4Results of the study on the effect of overexpressing the LOC339524 protein on cell migration ability in three types of cancer cells in Example 4 (pCAGGS: empty vector; pCAGGS-LOC-Flag: vector integrating SEQ ID NO.1; pCAGGS-0580-Flag: vector integrating SEQ ID NO.2).

[0024] Figure 5 Results of the study on the effect of overexpressing the LOC339524 protein on cell invasion ability in three types of cancer cells in Example 4 (pCAGGS: empty vector; pCAGGS-LOC-Flag: vector integrating SEQ ID NO.1; pCAGGS-0580-Flag: vector integrating SEQ ID NO.2). Detailed implementation manners

[0025] The present invention will be further described in detail below in conjunction with examples, but the implementation manners 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 can be obtained from commercial channels. 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 coding region sequence of LOC339524

[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 the expression vector

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

[0034] (1) Digestion of pCAGGS and the target gene: Take 1 μg of pCAGGS or the target gene, add 5 μl of 10× cutsmart buffer (BioLabs, SV0827), 1 μl of BstXI enzyme (Thermo Scientific TM , ER1021), 1 μl of BglII enzyme (Thermo Scientific TM , ER0081), add double-distilled water to make up to 50 μl, and digest at 37°C for 2 h. The digested products were detected by electrophoresis and recovered. The steps refer to the GeneJET Gel Extraction Kit (Thermo Scientific TM , K0691).

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

[0036] (3) Transformation:

[0037] Add the ligation product to be transformed into a tube containing TOP10 competent cells (Thermo Scientific TM, into the tube (50 μl of competent cells require 25 ng of DNA) of C404006. The volume should not exceed 5% of the competent cells. Gently rotate several times to mix the contents and incubate on ice for 30 min. Transfer the tube mixture into a circulating water bath heated to 42 °C and perform heat shock for 90 s without shaking the tube. Quickly transfer the tube to an ice bath to cool the cells for 1 min. Add 200 μl of SOC liquid medium to each tube, warm the medium to 37 °C using a water bath, then transfer the tubes to a shaker set at 37 °C and culture at 220 rpm for 45 min to allow the cells to recover and express the plasmid-encoded resistance marker gene. Transfer an appropriate volume (up to 200 μl for each 90 mm plate) of the transformed competent cells onto LB medium containing the corresponding antibiotic. Invert the plates and incubate at 37 °C. Colonies can appear after 16 hours.

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

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

[0040] MLARRDLGLVPHGVSGVSIAASSTPQGQAVCSPSVAAPSTLLLLRTHLLGAASLQGCGVLHILPIFLFSKGCRRDAQCACTVGPSASPRSGRGPGRGGGRRPRLGAARSGCPGAAAAGGPAVLHPWRRAGGRVRGASPPQGPQTARGFPLPSRWSSSPIPGCISIYPSPISFAHPGSLAPLGSPFPSPGPPSRSRLLCPGLRRGLTPGRWFRPDLGSLVTPRLLPLPNSGEPGIKPCAFLFFLLRAESTLHVCQGISSESERRTRSFFFFPRSCLL.

[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] MLARRDLGLVPHGVSGVSIAASSTPQGQAVCSPSVAAPSTLLLLRTHLLGAASLQGCGVLHILPIFLFSKGCRRDAQCACTVGPSASPRSGRGPGRGGGRRPRLGAARSGCPGAAAAGGPAVLHPWRRAGGRVRGASPPQGPQTARGFPLPSRWSSSPIPGCISIYPSPISFAHPGSLAPLGSPFPSPGPPSRSRLLCPGLRRGLTPGRWFRPDLGSLVTPRLLPLPNSGEPGIKPCAFLFFLLRAESTLHVCQGISSESERRTRSFFFFPRSCLL DYKDDDDK 。

[0043] Example 3: Transfection of Expression Vector

[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 5 cells / ml and seeded into 6-well plates at 2 ml per well using complete medium (Hyclone, SH30022.01) containing 10% fetal bovine serum (Lonsera, S711-0011S). After culturing for 24 h, the cell confluence was 70 - 80%. 3 μL of PolyJetTM (SignaGen, SL100489), 1 μg of pCAGGS-0580-Flag or pCAGGS were separately diluted in 200 μl of serum-free medium, gently mixed, and left standing at room temperature for 5 min. The serum-free medium containing PolyJetTM was added to the serum-free medium containing pCAGGS-0580-Flag or pCAGGS, and left standing at room temperature for 15 min (not exceeding 30 min) to obtain a transfection mixture. During the waiting process of the above steps, the medium in the 6-well plates was aspirated, 2 ml of 1×PBS was added for gentle washing, and after aspiration, 1.6 ml of fresh complete medium was added. After the standing at room temperature ended, the transfection mixture was added to the 6-well plates and placed in a cell culture incubator. After 24 h of transfection, pCAGGS-0580-Flag-transfected cells and pCAGGS-transfected cells were obtained for subsequent experiments.

[0045] The cells transfected for 24 h above (pCAGGS-0580-Flag-transfected cells and pCAGGS-transfected cells) were subjected to Western Blot detection to evaluate the protein expression level of LOC3339524. The specific steps are as follows:

[0046] (1) Total Protein Extraction and Quantification

[0047] Aspirate the medium in the 6-well plate, add 2 ml 1× ice-cold PBS and wash gently, repeat once. After aspirating PBS, add 100 μl RIPA lysis buffer (containing 1 μl PMSF) and lyse on ice for 30 min. Transfer the lysate containing cells to a 1.5 ml EP tube and centrifuge at 4°C, 12000 rpm / min for 15 min. Gently aspirate the supernatant, which is the total cell protein, and store at -80°C.

[0048] (2) Protein quantification

[0049] The total cell protein was quantified using the BCA protein concentration assay kit (Biyuntian, P0009). Take 1.2 ml of the protein standard preparation solution and add it to a tube of protein standard (30 mg BSA). After fully dissolving, prepare a 25 mg / ml protein standard solution. Take an appropriate amount of 25 mg / ml protein standard and dilute it to a final concentration of 0.5 mg / ml. For example, take 20 μl of 25 mg / ml protein standard and add 980 μl of diluent to prepare a 0.5 mg / ml protein standard. According to the number of samples, prepare the BCA working solution at a ratio of BCA reagent A: reagent B = 50:1 and mix thoroughly. Note: The BCA working solution is stable at room temperature for 24 hours. Add 0, 1, 2, 4, 8, 12, 16, 20 μl of the standard to the standard wells of the 96-well plate, and add the standard diluent to make up to 20 μl, which is equivalent to the standard concentration of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg / ml. Add an appropriate volume of sample to the sample well of the 96-well plate. If the sample is less than 20 μl, add the standard diluent to make up to 20 μl. Add 200 μl of BCA working solution to each well and place it at 37°C for 30 minutes. Use an enzyme marker to measure the absorbance of the wavelength of A562. Calculate the protein concentration of the cell based on the standard curve and the sample volume used, and quantify it to 5 μg / μl. Add 5×SDS-PAGE protein loading buffer (Biyuntian, P0285) to the cell protein at a volume of 1:4, mix well, boil in boiling water for 15 minutes, and store at -20°C.

[0050] (3) Western Blot

[0051] Glue making: Two clean glass plates are fixed on the glue making tank, and ddH 2 Observe the leakage. If there is no leakage, use filter paper to remove the ddH 2 O was sucked dry and ready for gel casting. According to the molecular weight of the protein, according to the instructions of the gel preparation kit (Biyuntian, P0012AC), the corresponding volume of 10% separation gel (7mL / block) and 5% concentration gel (2mL / block) were prepared. After the concentration gel was poured, the sample comb was inserted immediately, and it was careful not to generate bubbles. Let it stand at room temperature for 20 minutes and prepare for sample loading.

[0052] Sample loading: Pour freshly prepared 1× electrophoresis buffer to submerge the sample wells. Load samples sequentially using a micropipette, 20 μg for each sample.

[0053] Electrophoresis: Connect the positive and negative electrodes. Run at a constant voltage of 80 V for the stacking gel and 120 V for the separating gel. Stop electrophoresis when the target band runs to the middle of the separating gel according to the colored prestained protein marker (Thermo Scientific TM , 26616), and prepare for membrane transfer.

[0054] Membrane transfer: Cut the whole separating gel and cut the PVDF membrane according to the size of the separating gel. Activate the PVDF membrane (Millipore, GVWP04700) with methanol for 20 s before assembling the "sandwich". Assemble the "sandwich" in the order of thick filter paper, PVDF membrane, separating gel, filter paper, and sponge (no bubbles between the PVDF membrane and the separating gel). Place it in the membrane transfer apparatus according to the color and perform wet transfer at a constant current of 200 mA for 150 min.

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

[0056] Incubation with primary antibody: Wash the PVDF membrane once with TBST. Cut out the target band according to the colored prestained protein marker and place it in an antibody incubation box containing diluted Flag primary antibody (CST, 14793S) and β-actin (Wuhan Sanying, 66009-1-Ig), and incubate overnight at 4℃.

[0057] Incubation with secondary antibody: Take out the band incubated with the primary antibody yesterday, wash it 4 times with TBST on a shaker at room temperature for 5 min each time. After rinsing, place the band in an antibody incubation box containing diluted secondary antibody (Wuhan Sanying, SA00001-4) and incubate for 2 h at room temperature.

[0058] Development: Mix solution A and solution B in the ECL kit (Chongqing Baoguang) at a ratio of 1:1 in a brown glass bottle. Place the band on the lid of a well plate covered with sealing film, add the developing solution, develop and expose to obtain an image. Analyze the gray value of the band using ImageJ software.

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

[0060] Example 4: Cell phenotype after high expression

[0061] (1) Proliferation experiment

[0062] After trypsin digestion, the cells were collected by centrifugation at 800 rpm for 5 min, seeded at 1500 cells / well in a 96-well plate at 100 μl / well. After 24 h, when the cell confluence was about 20%, transfection of pCAGGS-0580-Flag or pCAGGS (0.1 μg / well) was started. The transfection method is referred to Example 3. The general process is as follows: Prepare the transfection mixture according to the ratio in Example 3, and add 100 μl of the transfection mixture to each well of the 96-well plate. Observe the cell growth status after transfection of pCAGGS-0580-Flag or pCAGGS under an inverted optical microscope. Place the cells under an inverted optical microscope for observation and take pictures. Cck8 was used to evaluate the number of cells obtained after 24 h, 48 h, and 72 h of transfection.

[0063] Prepare cck8 solution (Beyotime, C0041) at 100 μl / well, with the volume ratio of cck8: serum-free medium = 1:9. Aspirate the medium, add 100 μl of 1×PBS and wash gently, then aspirate and add the prepared cck8 solution, and incubate at 37 °C for 1 h. Measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader to evaluate the number of cells.

[0064] The results of the proliferation experiment are as Figure 3 shown. Figures A-C are the pictures taken, and Figures D-F are the statistical charts of the cck8 detection results. The results show that: compared with the cells transfected with pCAGGS (labeled as pCAGGS in the figure), the cells transfected with pCAGGS-0580-Flag (labeled as pCAGGS-0580-Flag in the figure) showed a significant decrease in proliferation at 48 h, and the difference was statistically significant (P < 0.01), indicating that transfection with pCAGGS-0580-Flag inhibited cell proliferation. It shows that overexpression of the LOC339524 protein in cancer cells has the effect of inhibiting cancer cell proliferation.

[0065] (2) Scratch assay

[0066] The cells obtained after 24 h of transfection (cells transfected with pCAGGS-0580-Flag and cells transfected with pCAGGS) were used for the experiment. When the cell confluence reached 99-100%, a "cross" scratch was made perpendicular to the bottom of the well plate with a 200 μl pipette tip, with uniform force, and try to ensure that the width was uniform and the scratch was horizontal and vertical. Wash the cells 3 times with PBS, add serum-free medium, and take pictures under an inverted optical microscope at 0 h, 24 h, 48 h, and 72 h. Among them, the migration rate and the proportion of the migration area were calculated by the following method:

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

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

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

[0070] The results of the migration experiment are as Figure 4 shown. Figures A-C are the photographed images, and Figures D-F are the statistical charts of the migration area ratio. The results show that: compared with the cells transfected with pCAGGS (labeled as pCAGGS in the figure), the migration rates of the cells transfected with pCAGGS-0580-Flag (labeled as pCAGGS-0580-Flag in the figure) were significantly decreased at 24h and 48h, and the difference was statistically significant (P<0.01), indicating that the transfection of pCAGGS-0580-Flag inhibited cell migration. It shows that the overexpression of LOC339524 protein in cancer cells has the effect of inhibiting cancer cell migration.

[0071] (3) Invasion experiment

[0072] The cells obtained after 24h of transfection (the cells transfected with pCAGGS-0580-Flag and the cells transfected with pCAGGS) were used for the experiment. At 4°C (operating on ice), Matrigel (BD, 356237) was diluted 1:8 with serum-free medium. 60 μl was taken and evenly added to the upper chamber surface of the bottom membrane of the Transwell chamber (Corning, 3422), and incubated in a 37°C incubator for 3h. After incubation, the excess liquid in the upper chamber was aspirated. 100 μl of serum-free medium was added to each well and placed in the incubator for 30 min. After trypsin digestion, the cells were collected by centrifugation at 800 rpm for 5 min, the supernatant was discarded, and the cells were gently washed twice with PBS, and then suspended in serum-free medium to adjust the cell density to 2.5×10 5 cells / ml. 500 μL of complete medium was added to the lower chamber of a 24-well plate, and then the Transwell chamber was placed in the 24-well plate with forceps. 200 μL of the cell suspension was taken and added to the upper chamber, and finally placed in the incubator for 24h. The chamber was taken out with forceps, the medium in the upper chamber was blotted dry, and the Matrigel and the cells in the upper chamber were gently wiped with a cotton swab. A new 24-well plate was taken and 500 μL of 4% paraformaldehyde (Beyotime, P0099) was added, and the chamber was placed in and fixed at room temperature for 30 min. The chamber was taken out, the fixing solution in the upper chamber was blotted dry, and transferred to a well containing 800 μl of 1% crystal violet (Solarbio, G1062) for staining for 30 min. After staining, the upper side of the chamber was gently wiped with a cotton swab to wipe off the dye non-specifically bound to the upper surface of the chamber.

[0073] 8) Gently wash and soak with PBS several times, 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 take pictures under an inverted microscope.

[0074] The invasion assay results were as follows Figure 5 As shown, Figure A is a photographic image, and Figure B is a statistical image of invasive cells. The results show that the number of cells transfected with pCAGGS (marked as pCAGGS in the figure) and cells transfected with pCAGGS-0580-Flag (marked as pCAGGS-0580-Flag in the figure) that passed through the matrix gel and basement membrane at 24 hours was significantly reduced, and the difference was statistically significant (P<0.01), indicating that cells transfected with pCAGGS-0580-Flag can inhibit cell invasion. This shows 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 tried to express the gene of SEQ ID NO.1 in cells. The LOC339524 gene is a human gene, and the cells that overexpress the gene in this scheme are also human cells. In theory, the gene of SEQ ID NO.1 can also be well expressed in human cells. Generally speaking, the reason for codon optimization is the preference of codons. Codon optimization is a process of improving gene expression and improving the translation efficiency of target genes by adapting to the codon preference of the host organism. Codon preference specifically refers to: different organisms have different frequencies of use of degenerate codons. There are fewer tRNAs corresponding to rare codons in cells, and the gene translation process that uses these codons at a high frequency is easily blocked, greatly reducing the protein expression level. If the frequency of use of synonymous codons in the gene matches that of the expression host, the expression level of the protein will be significantly improved. However, this scheme does not have the problem of codon preference between different species. It is the expression of human genes in human cells, so there is no current guiding principle 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 Figures 2 - 5pCAGGS-LOC-Flag. The preparation method of pCAGGS-LOC-Flag is the same as that of pCAGGS-0580-Flag in Example 3, and 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 vector containing SEQ ID NO.1 has a low expression level in cells, and its effect of inhibiting tumor proliferation, migration and invasion is poor. Although the human gene is expressed in human cells in this scheme, however, by converting the sequence of SEQ ID NO.1 into the sequence of SEQ ID NO.2 to express the same protein, the protein expression level can be greatly improved in human cells and a better cancer cell inhibition effect can be achieved, which is unexpected by the inventor. The use of the SEQ ID NO.2 sequence has achieved an unexpected technical effect.

[0078] The above are only the embodiments of the present invention, and common knowledge such as the specific structures and characteristics known in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the structure of the present invention, several deformations and improvements can still be made, and these should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicality of the patent. The protection scope required by this application should be based on the content of its claims, and the specific implementation manners described in the specification can be used to explain the content of the claims.

Claims

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

2. The use of the LOC339524 gene and its encoded protein in the preparation of a drug for treating cancer according to claim 1, 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.

3. The use of the LOC339524 gene and its encoded protein in the preparation of a drug for treating cancer according to claim 2, characterized in that: The amino acid sequence of the LOC339524 protein is shown in SEQ ID NO.3 or SEQ ID NO.

4.

4. Use of the LOC339524 gene and its encoded protein in the preparation of a drug for treating cancer according to any one of claims 1 to 3, characterized in that: The cancer is lung cancer, liver cancer or glioma.

5. The use of the LOC339524 gene and the protein encoded therein in the preparation of a drug for treating cancer according to claim 4, characterized in that: The lung cancer is non-small cell lung cancer, the liver cancer is hepatocellular carcinoma, and the glioma is glioblastoma.

6. Use of the LOC339524 gene and the protein encoded therein according to any one of claims 1 to 3 in the preparation of a drug for treating cancer, characterized in that: The amount of LOC339524 gene or LOC339524 protein is increased in cancer cells.

7. The use of the LOC339524 gene and the protein encoded therein in the preparation of a drug for treating cancer according to claim 6, characterized in that: The amount of the LOC339524 gene or LOC339524 protein in cancer cells is increased in an overexpressed manner.

8. The use of the LOC339524 gene and its encoded protein in the preparation of a drug for treating cancer according to claim 7, characterized in that: The overexpression method is: the cancer cells are transfected with an expression vector, and the expression vector is integrated with the LOC339524 gene.

9. The use of the LOC339524 gene and the protein encoded therein in the preparation of a drug for treating cancer according to claim 8, characterized in that: 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.

10. Use of the LOC339524 gene and the protein encoded therein according to any one of claims 1 to 3 in the preparation of a drug for treating cancer, characterized in that: The drug is used to inhibit cancer cell proliferation, migration and invasion.

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