Shear variant of LAG3 and application thereof

Abstract

The invention relates to the technical field of bioengineering, in particular to an LAG3 shear variant and application thereof. The nucleotide sequence of the splicing variant of the LAG3 provided by the embodiment of the invention is as shown in SEQ ID No. 1. Compared with an existing shear variant, the LAG3 new shear variant provided by the invention can better promote proliferation of a hepatoma cell line, and inhibition of expression of the LAG3 new shear variant can remarkably reduce growth of the hepatoma cell line, so that the specificity of a coding region of the LAG3 new shear variant has a wide application prospect in the aspects of diagnosis and treatment of hepatoma.

Description

Technical Field

[0001] The present application belongs to the field of bioengineering technology, and in particular relates to a splicing variant of LAG3 and its application. Background Art

[0002] Liver cancer is a malignant tumor disease that occurs in the liver and is characterized by high morbidity and mortality. Hepatocellular carcinoma (HCC) is a common pathological type of primary liver cancer. Studies have shown that genetic changes (including DNA gain and loss) as well as mutations and epigenomic changes have been identified as important factors in the occurrence of liver cancer; and many genetic pathways are dysregulated in the carcinogenic process.

[0003] LAG3 stands for Lymphocyte Activation Gene-3, also known as CD223. It is an immune checkpoint receptor protein that negatively regulates T cell function and is a member of the immunoglobulin superfamily. Currently, research on LAG3 mainly focuses on immune cells. It has also been reported that LAG3 is expressed in a variety of tumors, such as renal clear cell carcinoma (KIRC), gastric cancer, breast cancer, B cell lymphoma and lung cancer, but there are fewer studies on liver cancer. Summary of the invention

[0004] The purpose of this application is to provide a LAG3 splice variant and its application, aiming to solve the technical problems of providing new liver cancer targets and better coping with liver cancer treatment.

[0005] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:

[0006] In a first aspect, the present application provides a splice variant of LAG3, the nucleotide sequence of the splice variant is shown in SEQ ID No.1.

[0007] In a second aspect, the present application provides an application, namely, the use of a substance that inhibits the expression of a splice variant of LAG3 in the preparation of a drug for treating liver cancer, wherein the nucleotide sequence of the splice variant is shown in SEQ ID No.1.

[0008] The first aspect of the present application provides a newly discovered splice variant of LAG3, which is expressed in liver cancer cell lines and is different from the three major splice variant sequences currently reported. The present application further finds through cell biological functional studies that it can promote the proliferation of liver cancer cell lines more than existing splice variants, and inhibiting its expression can significantly reduce the growth of liver cancer cell lines. Therefore, the specificity of its coding region has broad application prospects in the diagnosis and treatment of liver cancer.

[0009] The application provided in the second aspect of the present application is based on the fact that inhibiting the expression of the newly discovered splice variant of LAG3 in the examples of the present application can significantly reduce the growth of liver cancer cell lines. Therefore, the substance that inhibits the expression of the splice variant of LAG3 can be used to prepare drugs for treating liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0011] Figure 1 This is a schematic diagram of Pre-mRNA splicing of the splicing variant of LAG3 in the embodiment of the present application;

[0012] Figure 2 is a comparison diagram of the splice variant of LAG3 in the embodiment of the present application and other variants;

[0013] Figure 3 This is a graph showing the PCR results of extracting the splice variant of LAG3 in the embodiment of the present application;

[0014] Figure 4 This is a diagram showing the drug resistance results of various stable cell lines of SMMC-7721 in the present application example;

[0015] Figure 5 This is a graph showing the drug resistance results of knocking down LAG3 in the SMMC-7721 stable cell line in the example of the present application. DETAILED DESCRIPTION

[0016] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0017] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0018] In the present application, "at least one" means one or more, "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0019] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0020] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0021] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass described in the embodiment description of the present application can be a mass unit known in the chemical industry such as μg, mg, g, kg, etc.

[0022] The terms "first" and "second" are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0023] In the examples of the present application, a new splice variant of LAG3 was discovered for the first time, which was expressed in liver cancer cell lines and was different from the three main splice variant sequences reported previously. Subsequent cell biological functional studies found that it was more able to promote the proliferation of liver cancer cell lines, indicating that it plays an important role in the occurrence and development of liver cancer, and the particularity of its coding region has broad application prospects in the diagnosis and treatment of liver cancer. Therefore, the examples of the present application provide the following technical solutions.

[0024] In a first aspect, the present invention provides a splice variant of LAG3. The nucleotide sequence of the splice variant of LAG3 provided in the present invention is shown in SEQ ID No.1.

[0025] SEQ ID No.1:

[0026]

[0027] The schematic diagram of the Pre-mRNA splicing of the LAG3 splicing variant is shown in Figure 1 As shown in the figure: LAG3 usually has 8 exons: E1, E2, E3, E4, E5, E6, E7, and E8; however, after splicing, the splice variant of the present application lacks exon 3 E3, and an intron is inserted between exon 1 E1 and exon 2 E2, resulting in a backward shift of the coding region. Compared with the three human LAG3 splice variants reported so far, Figure 2 As shown, the three transcripts of LAG3 in the human body currently included in the NCBI database are: LAG3 Tv1 (LAG3 Transcript variant1-1976 bp), LAG3 Tv2 (LAG3 Transcript variant2-2062 bp), and LAG3 Tv3 (LAG3 Transcript variant3-1733bp). LAG3 has four immunoglobulin domains (D1-D4) outside the membrane, of which Tv3 lacks exon 6 and the D4 structure. Currently, most of the research on LAG3 only focuses on its LAG3 Tv1 as an immune checkpoint on the surface of the immune cell membrane to play an immunosuppressive role. LAG3Δ3 is a splice variant of the nucleotide sequence newly discovered in the embodiment of the present application as shown in SEQ ID No.1.

[0028] Specifically, the amino acid sequence of the protein encoded by the splice variant of LAG3 provided in the examples of the present application is shown in SEQ ID No. 2.

[0029] SEQ ID No.2:

[0030] MDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQER FVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSSPGAQRSGRAPGALPAGHLLLFLILGVLSLLLLVTGAFGFHLWRRQWRPRRFSALEQGIHPPQAQSKIEELEQEPEPEPEPEPEPEPEPEPEQL.

[0031] In a second aspect, the present application provides an application, that is, the application of a substance that inhibits the expression of a splice variant of LAG3 in the preparation of a drug for treating liver cancer, wherein the nucleotide sequence of the splice variant is shown in SEQ ID No.1.

[0032] Based on the fact that inhibiting the expression of the newly discovered splice variant of LAG3 in the examples of the present application can significantly reduce the growth of liver cancer cell lines, the substance that inhibits the expression of the splice variant of LAG3 can be used to prepare drugs for treating liver cancer.

[0033] In some embodiments, the substance that inhibits the expression of LAG3 splice variants includes at least one of a protein, a polypeptide, and a small molecule compound that inhibits the activity of the protein encoded by the splice variant of LAG3. These substances can bind to the protein encoded by the splice variant whose amino acid sequence is shown in SEQ ID No. 2, and can degrade or reduce its activity, thereby reducing the growth of liver cancer cells.

[0034] In some embodiments, the substance that inhibits the expression of a splice variant of LAG3 includes a substance that knocks down the splice variant of LAG3 or a substance that knocks out the splice variant of LAG3.

[0035] The substance that knocks down the splice variant of LAG3 can be any substance that can make the gene with a nucleotide sequence as shown in SEQ ID No.1 difficult to express, such as a substance that silences the splice variant gene. For example, the substance that knocks down the splice variant includes at least one of miRNA, siRNA, dsRNA, and shRNA that silences the splice variant gene. Exemplary is a knockdown plasmid containing shRNA, wherein the shRNA sequence is: 5'-CGTCTCCATCATGTATAACttcaagagaGTTATACATGATGGAGACGttttttta-3'.

[0036] The substance that knocks out the splice variant of LAG3 can be a substance that achieves in any way that the human body or liver cancer cells do not produce the functional protein product of the splice variant of LAG3 (as shown in SEQ ID No. 2), such as removing all or part of the coding gene sequence, introducing a frameshift mutation so that no functional protein is produced, removing or changing the regulatory component (such as promoter editing) so that the coding gene sequence is not transcribed, and preventing translation by binding to mRNA. Usually, the knockout is performed at the genomic DNA level so that the offspring of the cell also permanently carry the knockout. For example, the substance that knocks out the splice variant includes a CRISPR / Cas9 gene editing system or a recombinant plasmid that knocks out the splice variant.

[0037] In some embodiments, the liver cancer targeted by the drug for treating liver cancer is hepatocellular carcinoma.

[0038] In the present embodiment, stable cells overexpressing LAG3 and cell lines knocking down LAG3 expression of human liver cancer cell SMMC-7721 were prepared, and the IC50 value after drug administration was used to reflect the cell activity by using the MTT colorimetric method. The IC50 value is the half-inhibitory concentration, which indicates that when the drug concentration reaches a certain level, the target biological activity or growth rate can be inhibited by 50%, and it is a key parameter for measuring the inhibitory effect of drugs on specific cells or pathogens.

[0039] The examples of the present application show that after the splice variant LAG3Δ3 of the examples of the present application is overexpressed in SMMC-7721 cells, the IC50 value of the stably transfected cell line overexpressing LAG3Δ3 after drug addition is higher than that of other splice variants such as LAG3 Tv1, indicating that the drug resistance of liver cancer cells overexpressing LAG3Δ3 is enhanced and the liver cancer cell line is more proliferative; and after knocking down the expression of LAG3Δ3, the corresponding IC50 value after drug addition is lower, indicating that after reducing its expression by inhibitors related to LAG3Δ3 expression, the proliferation of liver cancer cell lines can be better inhibited.

[0040] In some embodiments, the drug for treating liver cancer also includes anti-liver cancer molecular targeted drugs, that is, a combination of an inhibitor related to LAG3Δ3 expression and a currently commonly used anti-liver cancer molecular targeted drug as a drug for treating liver cancer, wherein the anti-liver cancer molecular targeted drug may include sorafenib.

[0041] In some embodiments, the drug for treating liver cancer further comprises a pharmaceutically acceptable excipient. For example, the excipient comprises at least one of a pharmaceutically acceptable carrier, a diluent, a filler, a binder, a preservative, a lubricant, a dispersant, a flavoring agent, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, an antioxidant, a colorant, and a stabilizer. The dosage form of the drug comprises at least one of a powder, a granule, a tablet, and a capsule.

[0042] The following describes the invention in conjunction with specific embodiments.

[0043] Example 1 Splice variants of LAG3

[0044] 1. Extraction of RNA from Hep3B liver cancer cell line

[0045] 1.1) Homogenization: In a centrifuge tube containing Hep3B liver cancer cells, 5 to 10 × 10 6 1 ml RNAex Pro Reagent (a total RNA extraction reagent produced by Hunan Aikerui Biotechnology Co., Ltd.) was added to each liver cancer cell and mixed, and then 0.1 ml chloroform was added for every 0.5 ml RNAex Pro Reagent added, the tube cap was tightly closed, shaken for 15 seconds, and placed at room temperature for 5 minutes.

[0046] 1.2) Centrifugal separation: The above centrifuge tube was subjected to refrigerated centrifugation at 4°C and 12,000 rpm for 15 minutes. After centrifugation, the mixture was divided into three layers: the lower layer was an organic phenol-chloroform layer, the upper layer was a colorless water layer, and RNA was present in the upper water layer.

[0047] 1.3) Obtaining the precipitate: Transfer the aqueous layer after centrifugation to a clean centrifuge tube, add an equal volume of isopropanol, invert to mix, and place at room temperature for 10 minutes. Then centrifuge at 4°C and 12,000 rpm for 10 minutes. Discard the supernatant and you will see a gelatinous precipitate containing RNA at the bottom of the tube.

[0048] 1.4) Washing the precipitate: Add 75% ethanol solution to wash the precipitate, and add 1 ml of 75% ethanol solution for every 1 ml of RNAexPro Reagent. After adding the ethanol solution, centrifuge at 4°C and 7500 rpm for 5 minutes and discard the supernatant.

[0049] 1.5) Redissolution: Place the washed precipitate at room temperature to dry, then add 50-100 μl of DEPC water to fully dissolve the precipitate to obtain the RNA solution of Hep3B liver cancer cell line, which can be stored at -70°C for future use.

[0050] 2. Reverse transcription

[0051] 2.1) The extracted RNA solution was prepared into the following reaction system 1 according to the instructions of the Evo M-MLV RT kit (produced by Hunan Aikerui Biotechnology Co., Ltd.):

[0052] dNTP Mix 1 μl;

[0053] Oligo dT primer 0.5 μl;

[0054] RNA sample solution 1 μl;

[0055] RNase free Water 7.5μl.

[0056] The reaction system 1 was subjected to the following reaction procedure: 75°C, 5 min; 4°C, ∞; and then stored at low temperature.

[0057] 2.2) The reaction solution after the reaction of the reaction system is prepared into the following reaction system 2:

[0058] Reaction solution of reaction system 1: 10 μl;

[0059] 5xRtase Reaction Buffer 4μl;

[0060] Evo M-MLV RTase 0.5 μl;

[0061] RNase free Water 5.5μl.

[0062] The reaction system 2 was subjected to the following reaction procedure: 50°C, 90 min; 95°C, 15 min; 4°C, ∞; when the reaction was completed, a cDNA solution of RNA reverse transcription of the Hep3B liver cancer cell line was obtained.

[0063] 3. PCR

[0064] Nested PCR was used to amplify the target fragment, and the primers used were as follows:

[0065] The first round primers are:

[0066] F1 (SEQ ID No. 3): 5'-TGCCCAGACCATAGGAGAG-3';

[0067] R1 (SEQ ID No. 4): 5'-ACTGGGCTGCTGAGATCTG-3'.

[0068] The second round primers are:

[0069] F2 (SEQ ID No. 5):

[0070] 5'-aattGCTAGCGCCGCCACCATGTGGGAGGCTCAGTTCC-3';

[0071] R2 (SEQ ID No. 6): 5'-aattGCGGCCGCTCAGCTCCAGGTCAGAGCT-3'.

[0072] According to the instructions of the 2×Accurate Taq premix (containing dye) kit (produced by Hunan Aikerui Bioengineering Co., Ltd.), the nested PCR amplification system was prepared as follows:

[0073]

[0074] The reaction program of the nested PCR amplification system was as follows: 95°C, 2 min; 95°C, 30 s, 55°C, 45 s, 72°C, 1 min, 35 cycles; 72°C, 5 min; 4°C, ∞.

[0075] Electrophoresis diagram Figure 3 As shown, lane 5 is the target fragment amplified by nested PCR.

[0076] 4. Sequencing

[0077] The nested PCR products were sequenced, and the sequencing results were organized using Snapgene software. The obtained sequences were then compared with the NCBI Gene database LAG3 Transcript variant 1. As a result, a new splice variant was found: LAG3Δ3, which lacks exon 3 and has an intron inserted between exons 1 and 2, resulting in a backward shift of the coding region. The specific sequence is shown in SEQ ID No. 1.

[0078] Example 2 Drug resistance experiment

[0079] 1. Preparation of stable cells of human hepatoma cell line SMMC-7721

[0080] The SMMC-7721 cells in the exponential growth phase were transfected with the control group pcDNA3.1 plasmid, LAG3 Tv1 overexpression plasmid, and LAG3Δ3 overexpression plasmid, respectively; wherein, the control group pcDNA3.1 plasmid was purchased from Addgene, and the LAG3 Tv1 overexpression plasmid and the LAG3Δ3 overexpression plasmid were obtained by inserting the corresponding nucleotide sequences into the pcDNA3.1 plasmid, and synthesized by General Biotechnology (Anhui) Co., Ltd. After transfection of the above plasmids for 48 hours, the culture medium (DMEM culture medium containing 10% FBS and 1% double antibody) with a concentration of 600 μg / ml G418 (purchased from Biyuntian Biotechnology) was added to the untransfected SMMC-7721 cells, and the cells were cultured continuously for two weeks. After all the SMMC-7721 cells in the untransfected group were killed by the drug, the remaining cells in the transfected group proved that the stable transfected cells had been successfully screened.

[0081] 2. MTT colorimetric assay

[0082] The above SMMC-7721 stably transfected cells were inoculated in 96-well plates at a density of 3000 cells per well. After 24 hours, the cells were allowed to adhere to the wall, the original culture medium was removed, and the following experiment was performed.

[0083] 2.1) Three SMMC-7721 stable cells transfected with pcDNA3.1 plasmid (control group), LAG3 Tv1 overexpression plasmid, and LAG3Δ3 overexpression plasmid were added to medium containing different concentrations of Sorafenib (DMEM medium containing 10% FBS and 1% double antibody) and cultured for 48 hours. After the culture, 100 μl of 0.5% MTT solution was added, incubated at 37°C for 4 hours, the MTT solution was discarded, 100 μl of DMSO was added, and the cells were shaken for 2 minutes. The OD value of each well was detected at a multifunctional microplate reader at a wavelength of 490 nm. The experiment was repeated 3 times, with 5 replicate wells in each group.

[0084] The relative growth rate of each group is as follows: Figure 4 As shown: the horizontal axis is the concentration of sorafenib in the culture medium (in μM), and the vertical axis is the relative growth rate (in %). 7721-NC represents the control group, and its IC50 is 6.575 μM; 7721-LAG3Tv1 represents the LAG3Tv1 overexpression group, and its IC50 is 6.929 μM; 7721-LAG3Δ3 represents the LAG3Δ3 overexpression group, and its IC50 is 7.438 μM. The data show that the drug resistance of liver cancer cells overexpressing LAG3Δ3 is stronger than that of the LAG3Tv1 overexpression group, and the proliferation is faster.

[0085] 2.2) SMMC-7721 stable cells transfected with LAG3 Tv1 overexpression plasmid and LAG3Δ3 overexpression plasmid were added with control group shGFP plasmid and experimental group sh-LAG3 knockdown plasmid respectively; wherein, the control group shGFP plasmid was obtained by replacing the strong promoter CMV in pCDH-EF1-copGFP-T2A-Puro plasmid (purchased from Addgene) with promoter H1; and the experimental group sh-LAG3 knockdown plasmid was obtained by replacing the strong promoter CMV in pCDH-EF1-copGFP-T2A-Puro plasmid with H1, and inserting the shRNA sequence 5'-CGTCTCCATCATGTATAACttcaagagaGTTATACATGATGGAGACGttttttta-3'; (SEQ ID No. 7). After the culture medium was cultured for 24 hours, the culture medium containing different concentrations of sorafenib was replaced and cultured for 48 hours. After the culture was completed, 100 μl of 0.5% MTT solution was added, incubated at 37°C for 4 h, the MTT solution was discarded, 100 μl of DMSO was added, and the cells were shaken for 2 min. The OD value of each well was detected on a multifunctional microplate reader at a wavelength of 490 nm. The experiment was repeated 3 times, with 5 replicate wells in each group.

[0086] The relative growth rate of each group is as follows: Figure 5Shown: the horizontal axis is the concentration of sorafenib in the culture medium (in μM), and the vertical axis is the relative growth rate (in %). 7721w-LAG3Δ3-NC represents SMMC-7721 stable cells transfected with LAG3Δ3 overexpression plasmid and shGFP plasmid as the control group, and its IC50 is 9.139μM; 7721w-LAG3Δ3-sh represents SMMC-7721 stable cells transfected with LAG3Δ3 overexpression plasmid and sh-LAG3 knockdown plasmid group, and its IC50 is 8.081μM; 7721-LAG3Tv1-NC represents SMMC-7721 stable cells transfected with LAG3 Tv1 overexpression plasmid and shGFP plasmid as the control group, and its IC50 is 9.052μM; 7721w-LAG3 Tv1-sh represents SMMC-7721 stable cells transfected with LAG3 Tv1 overexpression plasmid and sh-LAG3 knockdown plasmid group, and its IC50 is 8.611μM. The data showed that compared with the knockdown of LAG3Tv1, the liver cancer cells after knockdown of LAG3Δ3 had lower drug resistance and were more able to inhibit proliferation after drug administration.

[0087] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A splice variant of LAG3, characterized in that: The nucleotide sequence of the splice variant is shown in SEQ ID No.

1.

2. The splice variant according to claim 1, characterized in that The amino acid sequence of the protein encoded by the splice variant is shown in SEQ ID No.

2.

3. Use of a substance that inhibits the expression of a splice variant of LAG3 in the preparation of a drug for treating liver cancer, wherein the nucleotide sequence of the splice variant is shown in SEQ ID No.

1.

4. The use according to claim 3, characterized in that The amino acid sequence encoded by the splice variant is shown in SEQ ID No.

2.

5. The use according to claim 3, characterized in that The substance that inhibits the expression of LAG3 splice variants includes at least one of a protein, a polypeptide and a small molecule compound that inhibits the activity of the protein encoded by the splice variant.

6. The use according to claim 3, characterized in that The substance that inhibits the expression of LAG3 splice variants includes a substance that knocks down the splice variant or a substance that knocks out the splice variant.

7. The use according to claim 6, characterized in that: The substance for knocking down the splice variant comprises at least one of miRNA, siRNA, dsRNA and shRNA that silences the splice variant.

8. The use according to claim 6, characterized in that: The material for knocking out the splice variant includes a CRISPR / Cas9 gene editing system or a recombinant plasmid for knocking out the splice variant.

9. The use according to any one of claims 3 to 8, characterized in that: The liver cancer is hepatocellular carcinoma.

10. The use according to any one of claims 3 to 8, characterized in that: The drug for treating liver cancer also includes an anti-liver cancer molecular targeted drug; and / or, the drug for treating liver cancer also includes a pharmaceutically acceptable excipient.

Images