A TNS1 gene enhancer and its application

Abstract

The present invention relates to a TNS1 gene enhancer and its application. The nucleotide sequence encoding the TNS1 gene enhancer is shown as SEQ ID No: 1 and is named eTNS1. The present invention provides a novel TNS1 gene enhancer eTNS1, and it is confirmed by a luciferase reporter system that the eTNS1 can significantly enhance the transcriptional activity of the TNS1 promoter in A549 cells that have undergone epithelial-mesenchymal transition. By modifying the eTNS1, the mRNA transcription level of TNS1 is reduced, thereby obtaining the effect of regulating the migration inhibition of NSCLC. In addition, a preparation method of a product for inhibiting the expression of the TNS1 gene is also provided, making it easy to achieve the regulation of NSCLC migration inhibition.

Description

Technical Field

[0001] The present invention relates to the field of genetic modification technology, and particularly relates to a TNS1 gene enhancer and its application. Background Art

[0002] Non-small cell lung cancer (NSCLC) is a serious lung disease, accounting for the majority of lung cancer patients, about 80% of all lung cancers. Its early symptoms include chest pain, bloody sputum, low fever, cough, etc., and the late symptoms are local symptoms such as dyspnea, cough, hemoptysis, and systemic symptoms such as fatigue, weight loss, and decreased appetite. The causes of the disease may be related to smoking, environmental impact, genetic factors, etc. Among them, epithelial-mesenchymal transition (EMT) is an important cause and phenotype of NSCLC metastasis. Through EMT, tumor cells acquire stronger migration and invasion abilities, and are more likely to detach from the primary site and metastasize to other sites. At the same time, EMT also leads to changes in the morphology, molecular markers, and functions of tumor cells, and these changes together constitute the evidence that EMT is an important cause and phenotype of NSCLC metastasis. Therefore, how to inhibit epithelial-mesenchymal transition has become a new topic for treating the metastasis and deterioration of non-small cell lung cancer.

[0003] Enhancers are an important class of cis-acting elements in gene expression regulation. They can enhance the transcriptional activity of promoters, thereby increasing the gene expression level. Enhancers are usually short DNA sequences that can be located upstream, downstream, or within the target gene, or even at other positions on the chromosome far from the target gene. Due to the higher-order structure of chromatin (such as looping, topologically associating domains, etc.), enhancers far from the gene can still approach and affect the transcription of the target gene in three-dimensional space. Therefore, enhancers can be located anywhere in the gene, even spanning multiple genes or chromosomal regions. Based on the uncertainty of enhancer location, the discovery of gene enhancers is a complex process that requires a combination of experimental and computational methods. Experimental methods include DNA methylase sensitivity assays, chromatin immunoprecipitation (ChIP), etc., and computational methods include large-scale genomic data comparison, software prediction function analysis, etc. However, the DNA methylase sensitivity assay has a long experimental period, high technical requirements, and is prone to false positives and false negatives; ChIP requires a series of cross-linking, lysis, immunoprecipitation, and DNA purification steps, with high technical requirements and high costs; both large-scale genomic data comparison and software prediction function analysis may have errors and misjudgments, and a large number of predicted "enhancers" are found not to be able to truly increase the gene expression level after experiments. Therefore, the discovery of enhancers by computational methods needs to be combined with experimental verification to confirm.

[0004] Compared with the editing therapy targeting coding genes, editing enhancers can maintain the sequence of coding genes unchanged, with higher safety and clinical application potential. The first gene editing therapy approved by the FDA is to treat diseases by editing enhancer sequences that can regulate the expression of target genes. Finding and identifying functional enhancers that can truly inhibit the migration ability of non-small cell lung cancer cells is a hot topic and a challenge in the field of gene editing for the treatment of non-small cell lung cancer metastasis. Summary of the Invention

[0005] The purpose of the present invention is to disclose a TNS1 gene enhancer and its application, so as to solve one or more technical problems existing in the prior art and provide at least one beneficial choice or create conditions.

[0006] In the first aspect of the present invention, a tensin 1 (TNS1) gene enhancer, named eTNS1, is provided.

[0007] In the second aspect of the present invention, an sgRNA for targeted knockout of the TNS1 gene enhancer described in the first aspect of the present invention is provided.

[0008] In the third aspect of the present invention, the application direction of the TNS1 gene enhancer described in the first aspect of the present invention is provided.

[0009] In the fourth aspect of the present invention, a vector containing the sgRNA described in the second aspect of the present invention is provided.

[0010] In the fifth aspect of the present invention, a drug containing the sgRNA described in the second aspect of the present invention or the vector described in the fourth aspect of the present invention is provided.

[0011] The nucleotide sequence encoding the TNS1 gene enhancer described in the first aspect of the present invention is shown in SEQ ID No: 1.

[0012] The sgRNA described in the second aspect of the present invention includes sgRNA1 and / or sgRNA2; the nucleotide sequence encoding sgRNA1 is shown in SEQ ID No: 2; the nucleotide sequence encoding sgRNA2 is shown in SEQ ID No: 3.

[0013] The application described in the third aspect of the present invention is to regulate non-small cell lung cancer cells using the eTNS1.

[0014] In some application embodiments of the third aspect of the present invention, regulating non-small cell lung cancer cells is achieved by genetically modifying the eTNS1. Further, the genetic modification is insertion or deletion.

[0015] In some application embodiments of the third aspect of the present invention, the regulation is to reduce the transcription of TNS1 mRNA in the non-small cell lung cancer cells.

[0016] In some application embodiments of the third aspect of the present invention, the regulation is to inhibit the migration of the non-small cell lung cancer cells.

[0017] The vector described in the fourth aspect of the present invention contains the nucleotide sequence of the sgRNA described in the second aspect of the present invention.

[0018] The drug described in the fifth aspect of the present invention contains the sgRNA described in the second aspect of the present invention or the vector described in the fourth aspect of the present invention.

[0019] In some application embodiments of the fifth aspect of the present invention, the drug further includes pharmaceutically acceptable excipients.

[0020] The beneficial effects of the present invention are as follows:

[0021] 1. The present invention provides a new and truly functional TNS1 gene enhancer, and it is confirmed by the luciferase reporter system that eTNS1 can significantly enhance the transcription level of the TNS1 gene in A549 cells.

[0022] 2. By modifying the eTNS1, the reduction of the mRNA transcription of TNS1 can inhibit the migration ability of the cells undergoing EMT, and thus the effect of regulating the migration inhibition of NSCLC is obtained.

[0023] 3. A preparation method of a product for inhibiting the expression of the TNS1 gene is provided, making it easy to achieve the regulation of the migration inhibition of NSCLC. Description of the Drawings

[0024] Figure 1 is a schematic diagram of the eTNS1 plasmid described in Example 1;

[0025] Figure 2 is a bar chart of the relative luciferase activity in Example 1;

[0026] Figure 3 is a schematic diagram of constructing the eTNS1 knockout in Example 2;

[0027] Figure 4 is a bar chart of the relative expression level of the internal reference TUBB gene in Example 2;

[0028] Figure 5 is a schematic diagram of the wound closure experiment in Example 3

[0029] Figure 6 is the detection result of the metastasis of non-small cell lung cancer in vivo in Example 4;

[0030] Figure 7 It is the bar graph of the relative expression level of the TUBB gene in Example 5. Specific implementation manners

[0031] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, the modifications and substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0032] If not specifically specified, the technical means used in the examples are conventional means well-known to those skilled in the art.

[0033] For the molecular biology test methods not specifically described in the following examples, they are all referred to "Molecular Cloning: A Laboratory Manual" (Third Edition) or carried out according to the kits and product specifications; the biological materials of the kits can be obtained from commercial channels without special instructions.

[0034] Example 1, Identification of the enhancer of the TNS1 gene

[0035] Clone the nucleotide fragment of the eTNS1 downstream of the reporter gene luciferase with the ORI promoter, and detect whether the reporter gene has the function of the TNS1 gene enhancer. Specifically, it includes the following steps:

[0036] 1) Design PCR amplification primers according to the nucleotide sequence of the eTNS1. Forward primer eTNS1-F: 5’-TGGTAAAATCGATAAGGATCCtcaacttcaggggcctgctctgac-3’ (SEQ ID No:4); Reverse primer eTNS1-R: 5’-TCTCAAGGGCATCGGTCGACccaggaagccagaaggggaaagga-3’ (SEQ ID No:5).

[0037] 2) Extract the genomic DNA of A549 cells, and perform PCR amplification with the forward primer eTNS1-F and the reverse primer eTNS1-R.

[0038] 3) Transfer the amplification product into the pGL3-promoter vector using the BamH Ⅰ and Sal Ⅰ restriction enzyme sites, and the constructed eTNS1 plasmid is as Figure 1 shown.

[0039] 4) Induce A549 cells to undergo epithelial-mesenchymal transition (EMT) by adding TGFβ1 with a final concentration of 5 ng / mL to the culture medium.

[0040] 5) Transfect the constructed eTNS1 plasmid into A549 cells that have transformed into the mesenchymal state, and at the same time, the endogenous control vector pRL-TK is also transfected. After 48 hours of transfection, use the Dual Luciferase Assay Reagent (Vazyme) kit for detection.

[0041] The experimental results are as Figure 2 shown. After inserting the eTNS1 plasmid, compared with the blank control, the vector of the enhancer corresponding sequence showed significantly higher enhancer activity than the PGL3-promoter blank vector, and the activity was higher in A549 cells that had undergone mesenchymal transformation. It was proved that the eTNS1 has the function of the TNS1 gene enhancer.

[0042] Example 2, regulating NSCLC by knocking out the eTNS1

[0043] Use the system of transient transfection of Cas9 and sgRNA to knock out the TNS1 gene enhancer. The system includes a transient transfection plasmid that can transcribe sgRNA and simultaneously encode the Cas9 expression cassette and the puromycin resistance gene.

[0044] 1) Design sgRNA sequences targeting both ends of the TNS1 gene enhancer, namely sgRNA1 and sgRNA2. The nucleotide sequence encoding sgRNA1 is: 5'-ggtctgtgtagtgccgcggaagg-3' (SEQ ID No: 2); the nucleotide sequence encoding sgRNA2 is: 5'-catatgtacagactggaacaggg-3' (SEQ ID No: 3).

[0045] 2) Connect the nucleotide fragments shown in SEQ ID No: 2 and SEQ ID No: 3 to the transient transfection plasmid;

[0046] 3) Co-transfect the transient transfection plasmid obtained in step 2) into A549 cells in the cell proliferation phase;

[0047] 4) Screen positive cells by puromycin, pick monoclonal colonies, and perform genotype identification by genomic PCR. As Figure 3 shown, obtain the heterozygous knockout (HetroΔ) cell line and homozygous knockout (HomoΔ) cell line of eTNS1.

[0048] 5) The HetroΔ cell line and the HomoΔ cell line were subjected to epithelial-mesenchymal transition by adding 5 ng / mL of TGFβ1. After 48 hours of transformation, the expression level of the TNS1 gene was analyzed by real-time quantitative PCR amplification. mRNA of wild-type and different knockout genotypes (HetroΔ, HomoΔ) cells was extracted and reverse-transcribed into cDNA. After diluting the cDNA, it was used as a template and placed in a quantitative PCR instrument for reaction.

[0049] The detection results are as Figure 4 shown. Compared with the wild type, a cell line with inhibited enhancer expression was successfully constructed, and the mRNA expression level of the TNS1 gene showed a significant down-regulation of the transcription level, proving that the TNS1 gene enhancer has a significant regulatory effect on the TNS1 gene.

[0050] Example 3. Detection of the effect of eTNS1 on the migration ability of NSCLC cells in vitro

[0051] The wild-type A549 cells, the HetroΔ cell line, and the HomoΔ cell line were simultaneously induced to undergo epithelial-mesenchymal transition, and a scratch test was performed to observe whether the migration ability of NSCLC cells was also affected. The specific operation is as follows:

[0052] One day before the experiment, the unedited cell line, the HetroΔ cell line, and the HomoΔ cell line were inoculated into 96-well plates, about 30,000 cells per well, and 5 replicates were prepared for each cell to ensure that at least 3 replicates had uniform and clean scratches suitable for observation. On the day of the experiment, when it was observed that the cells just converged and spread out in a single layer, mitomycin with a final concentration of 2 μg / mL was added for 20 minutes to inhibit cell proliferation, and then uniform scratches were made. After washing away the floating cells, a medium containing 5 ng / mL TGFβ1 was added, and the cells were cultured in an Incucyte S3 instrument. Photos were taken every 2 hours starting from 15 minutes later until the scratches of the control group cells were closed (usually completely closed within 48 hours). ImageJ was used to analyze the closed area of different experimental groups for statistics. The results are as Figure 5 shown. The migration ability of the cells with eTNS1 knocked out was significantly decreased.

[0053] Example 4. Detection of the effect of eTNS1 on the in vivo invasion ability of NSCLC

[0054] The wild-type A549 cells stably expressing luciferase and the HomoΔ cell line were simultaneously induced to undergo epithelial-mesenchymal transition. Then, 150 μL of a PBS cell suspension containing 2 million wild-type or HomoΔ cells was injected into the tail vein of each nude mouse, and the luciferase substrate was injected once a week for in vivo imaging to observe the metastatic ability of the injected cells in vivo. The results are as Figure 6As shown, a large number of A549 cell residencies and proliferations were observed in the lungs of mice injected with wild-type A549 cells after 6 weeks, while the number of cancer cells in the lungs of mice injected with HomoΔ cells was significantly lower, indicating that compared with wild-type A549 cells, the ability of eTNS1 homozygous knockout cells to invade the lungs from blood vessels in vivo was significantly weakened. Therefore, the editing of the eTNS1 locus has the clinical treatment potential to inhibit the metastasis of non-small cell lung cancer.

[0055] Example 5: Interfere with eTNS1 using the dCas9-KRAB CRISPRi system to detect its effect on the expression of the TNS1 gene

[0056] The dCas9-KRAB system is a powerful tool for the transcriptional inhibition of endogenous genes. This technology relies on generating a Cas9 protein without nuclease activity. Amino acid mutations D10A and H840A are introduced into the two nuclease domains of Cas9, RuvC and NHN, respectively, so that the Cas9 protein loses its DNA cleavage activity but still retains the ability to bind DNA. By fusing and expressing KRAB to recruit suppressors to the Cas9 protein binding site, it is theoretically possible to achieve the effect of inhibiting the transcriptional activity of nearby genes without changing the DNA sequence. Therefore, it may replace CRISPR / Cas9 to knockout and interfere with the targeted gene and the cancer cell migration ability.

[0057] sgRNAs designed for the dCas9-KRAB system according to eTNS1 are sgRNA3 to sgRNA6 respectively; the nucleotide sequence encoding sgRNA3 is: 5’-gccacccaaagggaacagag-3’ (SEQ ID No: 6); the nucleotide sequence encoding sgRNA4 is: 5’-tctaggagccacctacccag-3’ (SEQ ID No: 7); the nucleotide sequence encoding sgRNA5 is: 5’-agactgcacagagctgtctg-3’ (SEQ ID No: 8); the nucleotide sequence encoding sgRNA6 is: 5’-gtgggaggaagtgatgtgct-3’ (SEQ ID No: 9).

[0058] Connect sgRNA3 to sgRNA6 into the dCas9-MeCP2-KRAB or dCas9-KRAB lentiviral vector respectively. Among them, dCas9-MECP2-KRAB is reported to be able to significantly enhance the inhibitory ability of KRAB. The obtained lentiviral vector is packaged in 293FT cells, and A549 cells are infected with the lentivirus solution secreted by the packaging cells. The results are as Figure 7As shown, sgRNA3 to sgRNA6 are all unable to down-regulate the expression of TNS1, indicating that the dCas9-KRAB system is difficult to replace Cas9 knockout to achieve the function of down-regulating the expression of TNS1.

[0059] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above-mentioned exemplary embodiments, and without departing from the spirit or basic characteristics of the present invention, the present invention can be implemented in other specific forms. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, it is intended to embrace all changes falling within the meaning and scope of the equivalent elements of the claims in the present invention.

Claims

1. TNS1 gene enhancer, characterized in that The nucleotide sequence encoding the TNS1 gene enhancer is shown in SEQ ID No:

1.

2. The sgRNA for targeted knockout of the TNS1 gene enhancer according to claim 1, characterized in that: Including sgRNA1 and / or sgRNA2; the nucleotide sequence encoding the sgRNA1 is shown as SEQ ID No: 2; the nucleotide sequence encoding the sgRNA2 is shown as SEQ ID No:

3.

3. Use of the sgRNA according to claim 2 in the preparation of a drug for treating non-small cell lung cancer.

4. The use according to claim 3, characterized in that: The sgRNA genetically modifies the TNS1 gene enhancer to achieve regulation of non-small cell lung cancer cells, and the nucleotide sequence encoding the TNS1 gene enhancer is shown in SEQ ID No:

1.

5. The application according to claim 4, characterized in that: The genetic modification is an insertion or a deletion.

6. The use according to claim 4, characterized in that: The regulation is to reduce the transcription of TNS1 mRNA in the non-small cell lung cancer cells.

7. The use according to claim 4, characterized in that: The regulation is to inhibit the migration of the non-small cell lung cancer cells.

8. A carrier, characterized in that Comprising a nucleotide sequence encoding the sgRNA according to claim 2.

9. A drug, characterized in that Containing the sgRNA according to claim 2 or the vector according to claim 8.

10. The drug according to claim 9, characterized in that: Pharmaceutically acceptable excipients are also included.

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