A splice variant of lag3 and uses thereof

Abstract

The application relates to the technical field of bioengineering, in particular to a LAG3 splicing variant and application thereof. The nucleotide sequence of the LAG3 splicing variant provided in the embodiment of the application is shown in SEQ ID No. 1. The LAG3 new splicing variant provided in the application can promote the proliferation of a hepatoma cell line more than the existing splicing variant, and the inhibition of the expression of the LAG3 new splicing variant can significantly reduce the growth of the hepatoma cell line, so that the particularity of the coding region has a broad application prospect in the diagnosis and treatment of hepatoma.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of bioengineering, and particularly relates to a splice variant of LAG3 and application thereof. BACKGROUND

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

[0003] LAG3, which stands for Lymphocyte Activation Gene-3, is also known as CD223, and is an immune checkpoint receptor protein. Its main function is to negatively regulate the function of T cells, and it belongs to the immunoglobulin superfamily member. At present, the related research on LAG3 mainly focuses on immune cells, and it is also reported that LAG3 is expressed in various tumors, such as renal clear cell carcinoma (KIRC), gastric cancer, breast cancer, B-cell lymphoma and lung cancer, but there are few related researches on liver cancer. SUMMARY

[0004] The present application aims to provide a splice variant of LAG3 and application thereof, and aims to solve the technical problems of providing a new liver cancer target and better coping with liver cancer treatment.

[0005] To achieve the above application purposes, the technical solutions adopted by the present application are as follows:

[0006] In a first aspect, the present application provides a splice variant of LAG3, and 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, i.e. the application of a substance for inhibiting the expression of a splice variant of LAG3 in the preparation of a liver cancer treatment drug, and 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 main splice variants reported at present. Further, through cell biological function research, it is found that the splice variant can promote the proliferation of liver cancer cell lines more than the existing splice variants, and the inhibition of its expression can significantly reduce the growth of liver cancer cell lines, so that the particularity of its coding region has a broad application prospect in the diagnosis and treatment of liver cancer.

[0009] The application provides the use of the substance for inhibiting the expression of the splice variant of LAG3, so that the growth of the hepatoma cell line is significantly reduced, and the substance for inhibiting the expression of the splice variant of LAG3 can be used for preparing a drug for treating hepatoma. BRIEF DESCRIPTION OF DRAWINGS

[0010] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments or prior art description will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0011] Figure 1 is a pre-mRNA splicing schematic diagram of the splice variant of LAG3 in the embodiments of the present application;

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

[0013] Figure 3 is a PCR result diagram of extracting the splice variant of LAG3 in the embodiments of the present application;

[0014] Figure 4 is a drug resistance result diagram of various stable cell lines of SMMC-7721 in the embodiments of the present application;

[0015] Figure 5 is a drug resistance result diagram of the stable cell line of SMMC-7721 knocking down LAG3 in the embodiments of the present application. DETAILED DESCRIPTION

[0016] In order to make the technical problems, technical solutions and beneficial effects to be solved in the present application more clear and obvious, the present application will be further described in detail in combination with 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 the present application, the term "and / or" describes the association relationship of the associated objects, which means that there can be three kinds of relationships, for example, A and / or B can mean that A exists alone, A and B exist together, and B exists alone. Wherein A and B can be singular or plural. The character " / " generally represents an "or" relationship between the associated objects before and after it.

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

[0019] It should be understood that the size of the sequence number of the above processes in various embodiments of the present application does not mean the order of execution, and part or all of the steps can be executed in parallel or in sequence, and the execution order of the processes should be determined according to 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 the plural forms, unless the context clearly indicates otherwise.

[0021] The weight of the related components mentioned in the specification of the embodiments of the present application can not only refer to the specific content of each component, but also represent the weight ratio relationship between each component, therefore, as long as the content of the related components in the specification of the embodiments of the present application is enlarged or reduced in proportion, it is within the scope disclosed in the specification of the embodiments of the present application. Specifically, the mass mentioned in the specification of the embodiments of the present application can be μg, mg, g, kg and other mass units commonly known in the chemical field.

[0022] The terms "first", "second" are only for the purpose of description, used to distinguish objects such as substances from each other, and cannot 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 can also be called the second XX, and similarly, the second XX can also be called the first XX. Therefore, the features limited by "first" and "second" can explicitly or implicitly include one or more of the features.

[0023] In the embodiments of the present application, a new splice variant of LAG3 is first discovered, which is expressed in hepatocellular carcinoma cell lines, and is different from the three main splice variant sequences reported in the prior art. Subsequent cell biological function research shows that it can better promote the proliferation of hepatocellular carcinoma cell lines, indicating that it plays an important role in the occurrence and development of hepatocellular carcinoma. The particularity of its coding region has broad application prospects in the diagnosis and treatment of hepatocellular carcinoma. Therefore, the embodiments of the present application provide the following technical solutions.

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

[0025] SEQ ID No. 1:

[0026]

[0027] The schematic diagram of the pre-mRNA splicing of the splicing variant of LAG3 is shown in Figure 1 Generally, LAG3 has 8 exons: E1, E2, E3, E4, E5, E6, E7, and E8; and by cutting and splicing, the splicing variant of the present application lacks the third exon E3, and a segment of intron is inserted between the first exon E1 and the second exon E2, resulting in the rearward shift of the coding region. Compared with the three human LAG3 splicing variants reported at present, as shown in Figure 2 LAG3 Tv1 (LAG3 Transcript variant 1-1976 bp), LAG3 Tv2 (LAG3 Transcript variant 2-2062 bp), and LAG3 Tv3 (LAG3 Transcript variant 3-1733 bp) in the NCBI database. LAG3 has four immunoglobulin domains (D1-D4) outside the membrane, and Tv3 lacks the D4 structure due to the lack of the sixth exon. At present, most of the researches on LAG3 only focus on the immune inhibitory effect of LAG3 Tv1 on the membrane surface of immune cells as an immune checkpoint. LAG3Δ3 is a newly discovered splicing variant with the nucleotide sequence as shown in SEQ ID No. 1.

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

[0029] SEQ ID No. 2:

[0030] MDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSSPGAQRSGRAPGALPAGHLLLFLILGVLSLLLLVTGAFGFHLWRRQWRPRRFSALEQGIHPPQAQSKIEELEQEPEPEPEPEPEPEPEPEPEQL.

[0031] In a second aspect, the embodiments of the present application provide an application. That is, the application of the substance for inhibiting the expression of the splice variant of LAG3 in the preparation of a drug for treating liver cancer. The nucleotide sequence of the splice variant is shown in SEQ ID No. 1.

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

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

[0034] In some embodiments, the substance for inhibiting the expression of the splice variant of LAG3 includes a substance for knocking down the splice variant of LAG3 or a substance for knocking out the splice variant of LAG3.

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

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

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

[0038] The present application embodiment reflects the cell activity by IC50 value after drug administration through MTT colorimetric method. IC50 value, i.e. half-inhibitory concentration, indicates that when the drug concentration reaches a certain degree, it can inhibit the target biological activity or growth rate by 50%, which is a key parameter for measuring the inhibitory effect of the drug on specific cells or pathogens.

[0039] The present application embodiment shows that after overexpression of the cleavage variant LAG3Δ3 of the present application embodiment in SMMC-7721 cells, the IC50 value of the stable cell line overexpressing LAG3Δ3 is higher than that of other cleavage variants such as LAG3 Tv1, indicating that the drug resistance of liver cancer cells overexpressing LAG3Δ3 is enhanced, and the liver cancer cell line can proliferate more; and after knocking down the expression of LAG3Δ3, the corresponding IC50 value after drug administration is lower, indicating that the liver cancer cell line can be more inhibited after reducing its expression by LAG3Δ3 expression-related inhibitors.

[0040] In some embodiments, the liver cancer treatment drug also includes an anti-liver cancer molecular targeted drug, i.e. a LAG3Δ3 expression-related inhibitor and a currently commonly used anti-liver cancer molecular targeted drug combination as a liver cancer treatment drug, wherein the anti-liver cancer molecular targeted drug can include sorafenib.

[0041] In some embodiments, the liver cancer treatment drug also includes a pharmaceutically acceptable excipient. For example, the excipient includes at least one of a pharmaceutically acceptable carrier, a diluent, a filler, a binder, a preservative, a lubricant, a dispersant, a flavoring and deodorizing agent, a humectant, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, an antioxidant, a coloring agent, a stabilizer. The dosage form of the drug includes at least one of a powder, a granule, a tablet, a capsule.

[0042] The following will be described in conjunction with specific embodiments.

[0043] Example 1 Cleavage variants of LAG3

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

[0045] 1.1) Homogenization: In a centrifuge tube containing Hep3B liver cancer cells, add 1 ml RNAex Pro Reagent (total RNA extraction reagent produced by Hunan Aikuo Biological Engineering Co., Ltd.) per 5-10 x 10 6 liver cancer cells, then add 0.5 ml RNAex Pro Reagent and 0.1 ml chloroform per 5-10 x 10

[0046] 1.2) Centrifugal separation: The centrifugal tube is centrifugally separated at 4℃, 12000 rpm for 15 minutes. After centrifugation, the mixture is divided into three layers: the lower layer is the organic phenol chloroform layer, the upper layer is the colorless water-like layer, and the RNA exists in the upper water-like layer.

[0047] 1.3) Obtain the precipitate: The water-like layer after centrifugal separation is transferred to a clean centrifugal tube, an equal volume of isopropanol is added, and after mixing, it is placed at room temperature for 10 minutes, and then centrifuged at 4℃, 12000 rpm for 10 minutes. After discarding the supernatant, a gelatinous precipitate containing RNA is seen at the bottom of the tube.

[0048] 1.4) Wash the precipitate: The precipitate is washed with a 75% ethanol solution, and for every 1 ml of RNAexPro Reagent used, 1 ml of 75% ethanol solution is added. After adding the ethanol solution, centrifuge at 4℃, 7500 rpm for 5 minutes, and discard the supernatant.

[0049] 1.5) Re-dissolve: After washing, the precipitate is placed at room temperature, dried, and then 50-100 μl of DEPC water is added to fully dissolve the precipitate, obtaining an RNA solution of the Hep3B liver cancer cell line, which can be stored at -70℃ for future use.

[0050] 2, Reverse transcription

[0051] 2.1) The extracted RNA solution is prepared according to the instructions of the Evo M-MLV RT kit (produced by Hunan Aikuo Biological Engineering Co., Ltd.) as follows:

[0052] dNTP Mix 1 μl;

[0053] Oligo dT primer 0.5 μl;

[0054] RNA sample solution 1 μl;

[0055] RNase free Water 7.5 μl.

[0056] The reaction system 1 is subjected to the following reaction program: 75℃, 5 min; 4℃, ∞; then store at low temperature.

[0057] 2.2) The reaction solution after reaction of the reaction system is prepared as follows:

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

[0059] 5xRtase Reaction Buffer 4 μl;

[0060] Evo M-MLV RTase 0.5 μl;

[0061] RNase free Water 5.5 μl.

[0062] Reaction system 2 was subjected to the following reaction procedure: 50℃, 90min; 95℃, 15min; 4℃, ∞; reaction ended, to obtain the cDNA solution of RNA reverse transcription of Hep3B hepatoma cell line.

[0063] 3. PCR

[0064] The target fragment was amplified by nested PCR, and the primers used were as follows:

[0065] The first round of primers were as follows:

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

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

[0068] The second round of primers were as follows:

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

[0070] 5'-aattGCTAGCGCCGCCACCATGTGGGAGGCTCAGTTCC-3';

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

[0072] The nested PCR amplification system was prepared according to the instructions of 2x Accurate Taq Premix (containing dye) kit (produced by Hunan Aikuo Biological Engineering Co., Ltd.):

[0073]

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

[0075] The electrophoretogram is shown in Figure 3 Lane 5 is the target fragment amplified by nested PCR.

[0076] 4. Sequencing

[0077] The obtained nested PCR product was sequenced, and the obtained sequencing results were arranged by Snapgene software, and then the obtained sequence was compared with LAG3 Transcript variant 1 in NCBI Gene database, and a new splice variant, LAG3Δ3, was found, which lacks the third exon and inserts an intron between the first and second exons, resulting in a shift of the coding region, and the specific sequence is shown in SEQ ID No. 1.

[0078] Example 2 Drug resistance experiment

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

[0080] SMMC-7721 cells in the exponential growth phase were transfected with 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, which was synthesized by General Biotech (Anhui) Co., Ltd. After 48 h of transfection of the above plasmids, another group of untransfected SMMC-7721 cells were added to the culture medium (DMEM culture medium containing 10% FBS and 1% double-antibiotic) with a concentration of 600 μg / ml G418 (purchased from Biyun Tian Bio) and continued to be cultured, and after two weeks of continuous screening, when the SMMC-7721 cells in the untransfected group were all 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 method

[0082] The above SMMC-7721 stable transfected cells were inoculated in a 96-well plate at a density of 3000 cells per well. After 24 h, the cells were adhered, the original culture medium was aspirated, and the following experiments were performed.

[0083] 2.1) The three kinds of SMMC-7721 stable transfected cells transfected with pcDNA3.1 plasmid (control group), LAG3 Tv1 overexpression plasmid and LAG3Δ3 overexpression plasmid were added to culture medium (DMEM culture medium containing 10% FBS and 1% double-antibiotic) containing different concentrations of Sorafenib for 48 h. After the culture was completed, 100 μl of 0.5% MTT solution was added, and after 4 h of incubation at 37°C, the MTT solution was aspirated, 100 μl of DMSO was added, and the OD value of each well was detected at a wavelength of 490 nm on a multifunctional enzyme label instrument. The experiment was repeated 3 times, and 5 replicate wells were set in each group.

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

[0085] 2.2) SMMC-7721 cells transfected with LAG3 Tv1 overexpression plasmid and LAG3Δ3 overexpression plasmid were added to both the control group shGFP plasmid and the experimental group sh-LAG3 knockdown plasmid. The control group shGFP plasmid was prepared by replacing the strong promoter CMV in the pCDH-EF1-copGFP-T2A-Puro plasmid (purchased from Addgene) with promoter H1. The experimental group sh-LAG3 knockdown plasmid was prepared by replacing the strong promoter CMV in the pCDH-EF1-copGFP-T2A-Puro plasmid with H1 and then inserting the shRNA sequence 5'-CGTCTCCATCATGTATAACttcaagagaGTTATACATGATGGAGACGtttttta-3' (SEQ ID No. 7). After culturing for 24 hours, the medium was replaced with a medium containing different concentrations of sorafenib, and culturing continued for 48 hours. After the culture was completed, 100 μl of 0.5% MTT solution was added, and the mixture was incubated at 37°C for 4 h. The MTT solution was then discarded, and 100 μl of DMSO was added. The mixture was shaken for 2 min, and the OD value of each well was measured using a multi-mode microplate reader at a wavelength of 490 nm. The experiment was repeated 3 times, with 5 replicates per group.

[0086] The final relative growth rate of each group is as follows: Figure 5The horizontal axis represents the concentration of sorafenib in the medium (unit: μM), and the vertical axis represents the relative growth rate (unit: %). 7721w-LAG3Δ3-NC represents SMMC-7721 stable cells transfected with LAG3Δ3 overexpression plasmid plus shGFP plasmid as a control group, and the IC50 thereof is 9.139 μM; 7721w-LAG3Δ3-sh represents SMMC-7721 stable cells transfected with LAG3Δ3 overexpression plasmid plus sh-LAG3 knockdown plasmid group, and the IC50 thereof is 8.081 μM; 7721-LAG3Tv1-NC represents SMMC-7721 stable cells transfected with LAG3 Tv1 overexpression plasmid plus shGFP plasmid as a control group, and the IC50 thereof is 9.052 μM; and 7721w-LAG3 Tv1-sh represents SMMC-7721 stable cells transfected with LAG3 Tv1 overexpression plasmid plus sh-LAG3 knockdown plasmid group, and the IC50 thereof is 8.611 μM. The data show that the drug resistance of liver cancer cells after knockdown of LAG3Δ3 is lower than that after knockdown of LAG3 Tv1, and the proliferation after administration is more inhibited.

[0087] The above only represents the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall 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 splicing variant is shown as SEQ ID No.

1.

2. The shear variant of claim 1, wherein, The amino acid sequence of the protein encoded by the splicing variant is shown as SEQ ID No.

2.

3. Use of a combination of a substance for inhibiting expression of a splicing variant of LAG3 and sorafenib in the preparation of a medicament for treating liver cancer, wherein the nucleotide sequence of the splicing variant is shown as SEQ ID No.

1. The substance is shRNA, and the nucleotide sequence of the shRNA is: 5'-CGTCTCCATCATGTATAACttcaagagaGTTATACATGATGGAGACGtttttta-3'; The liver cancer is hepatocellular carcinoma.

4. Use according to claim 3, characterized in that, The amino acid sequence of the protein encoded by the splicing variant is shown as SEQ ID No.

2.

5. Use according to any one of claims 3-4, characterized in that, The medicament for treating liver cancer further comprises a pharmaceutically acceptable excipient.

Images