MEG3-rat-448 sequence and application thereof

Abstract

The invention discloses an MEG3-rat-448 sequence and an application of the MEG3-rat-448 sequence. A primer sequence corresponding to the MEG3-rat-448 sequence is as shown in SEQ ID No. 1-2. The invention further discloses the application of the MEG3-rat-448 sequence in the aspect of regulating and controlling the intestinal Cajal cell function. The MEG3-rat-448 sequence is used for regulating intestinal movement, sensory function and endocrine activity by regulating intestinal Cajal cells. The invention also discloses an application of the MEG3-rat-448 sequence as an intestinal function regulation drug. The invention also discloses an application of the MEG3-rat-448 sequence in virus packaging.

Description

Technical Field

[0001] The present invention relates to the application field of MEG3-rat-448 sequence, and in particular to MEG3-rat-448 sequence and application thereof. Background Art

[0002] Intestinal Cajal cells, as an important component of the enteric nervous system, are responsible for regulating intestinal motility, secretion, and sensory functions, and play a key role in maintaining normal intestinal physiological functions. However, the dysfunction of intestinal Cajal cells is closely related to the pathogenesis of a variety of intestinal diseases. Therefore, the study of their functional regulation has important scientific significance and clinical value.

[0003] MEG3 (Maternally Expressed Gene 3) is a long non-coding RNA that has attracted widespread attention in the fields of biology and medicine in recent years. Many studies have shown that MEG3 has the ability to regulate multiple biological functions such as cell proliferation, differentiation, and apoptosis. Especially in terms of cell regulation, MEG3 shows unique advantages and can achieve precise regulation of cell functions by interacting with specific molecular targets. When exploring the regulatory effect of MEG3 on Cajal cells, we used quantitative real-time polymerase chain reaction (qPCR) technology to conduct in-depth research on different fragments of the MEG3 gene. After rigorous experimental design and data analysis, we found that the MEG3-rat-448 sequence showed the best effect when interfering with Cajal cells. This discovery not only reveals the specific regulatory effect of the MEG3-rat-448 sequence on Cajal cells, but also provides strong support for subsequent viral packaging and gene therapy research.

[0004] Based on the above findings, we decided to use the MEG3-rat-448 sequence for viral packaging to further explore its potential applications in Cajal cell function regulation and disease treatment. Summary of the invention

[0005] In order to solve the problems existing in the above-mentioned prior art, the purpose of the present invention is to provide a MEG3-rat-448 sequence and its application. By optimizing the virus packaging technology, the MEG3-rat-448 sequence is effectively delivered to Cajal cells, thereby achieving precise regulation of intestinal function. It is expected to open up new ways for the treatment of intestinal diseases and bring better treatment effects and quality of life to patients.

[0006] To achieve the above object, the technical solution of the present invention is:

[0007] MEG3-rat-448 sequence, the primer sequence corresponding to the MEG3-rat-448 sequence is shown in SEQ ID No.1-2.

[0008] Furthermore, the MEG3-rat-448 sequence is used to inhibit the function of intestinal Cajal cells.

[0009] Furthermore, the MEG3-rat-448 sequence regulates intestinal motility, sensory function and endocrine activity by regulating intestinal Cajal cells.

[0010] Furthermore, the MEG3-rat-448 sequence is used as a drug for regulating intestinal function.

[0011] Further, the application of the MEG3-rat-448 sequence in virus packaging

[0012] Further, the specific operation of the virus packaging of the MEG3-rat-448 sequence is as follows: according to the RNA sequence information of MEG3, PCR amplification primers are designed, and the upstream and downstream primers are respectively added with restriction sites XbaI and EcoRI and protective bases, and the upstream primer is added with a kozak sequence. PCR primer sequence: Forward primer-MEG3: 5'-TCTAGA (XbaI) GCCACC (kozak) AGCCCCTAGCGCAGA-3'; Revese primer-MEG3: 5'-GAATTC (EcoRI) TTGTTAAGACAGGAAACAC-3', and the PCR length is 1592bp. Real Time-PCR detection, primer sequences: Beta actin-forward primer, 5'-CCTGTACGCCAACACAGTGC-3', Beta acin-reverse primer 5'-ATACTCCTGCTTGCTGATCC-3', PCR product length is 211bp; MEG3-forward primer 5'-TGCGGATGGAAGCTGCGGAAGAG-3', MEG3-reverse primer 5'-CGCCCAAACCAGGAAGGAGACGAG-3', PCR product length is 102bp.

[0013] Compared with the prior art, the beneficial effects of the present invention are:

[0014] MEG3-rat-448 sequence and its application in virus packaging. First, the MEG3-rat-448 sequence has significant biological activity and can effectively interfere with the function of intestinal Cajal cells. This discovery reveals the potential role of the MEG3 gene in regulating intestinal Cajal cells and provides a new candidate gene fragment for subsequent gene therapy and cell regulation research.

[0015] Secondly, the MEG3-rat-448 sequence has the best interference effect on intestinal Cajal cells, indicating that it may have unique advantages in regulating intestinal function. As an important component of the intestinal nervous system, intestinal Cajal cells play a key regulatory role in intestinal motility, sensory function, and endocrine activity. Therefore, by regulating the function of intestinal Cajal cells, the MEG3-rat-448 sequence is expected to provide new strategies and methods for the treatment of intestinal diseases.

[0016] This result also proves the effectiveness and reliability of qPCR technology in gene expression analysis and functional screening. Through precise quantitative detection, we can evaluate the interference effects of different gene fragments on specific cell types, providing a strong basis for subsequent gene therapy and cell regulation research.

[0017] The best result of MEG3-rat-448 interfering with intestinal Cajal cells proved the potential role and value of this fragment in regulating intestinal function, and provided new ideas and methods for subsequent gene therapy and cell regulation research. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 These are bright-field images of intestinal cajal transfected with different NEG3 adenovirus fragments, showing the overall morphology of the cells;

[0019] Figure 2 Different fragments of NEG3 adenovirus were transfected into intestinal cajal. Bright field is the dark field cell image after fluorescence staining;

[0020] Figure 3 These are the bright field images of Cajal cells after empty vector transfection and the dark field images after fluorescent staining;

[0021] Figure 4 This is a bright field image of normal intestinal cajal.

[0022] Figure 5 The bar graph shows the relative transcripts (gene / GAPDH) of different MEG3 groups. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments:

[0024] like Figure 1-5 As shown,

[0025] Experimental example:

[0026] 1. Experimental instruments, consumables and reagents

[0027] 1.1 Experimental instruments and consumables

[0028] Table 1 Main experimental instruments and manufacturers

[0029]

[0030] 1.2 Experimental Reagents

[0031] Table 2 Main experimental reagents and manufacturers

[0032]

[0033] 2. Service content and process

[0034] Introducing the target nucleic acid into eukaryotic cells.

[0035] 3. Cell information and drug preparation

[0036] 3.1 Cell Information

[0037] Rat small intestinal cajal interstitial cell culture medium: Rat small intestinal cajal interstitial cell culture medium

[0038] 3.2 Drug preparation

[0039] The small fragments were prepared into 20 μM stock solution with DEPC water and stored at -20°C for later use.

[0040] 4 Experimental methods and procedures

[0041] 4.1 Cell passaging and expansion culture

[0042] When the cell density reaches 80%, the cells are passaged:

[0043] (1) Discard the culture medium and wash once with PBS;

[0044] (2) Add 1-2 ml of 0.25% trypsin to digest the cells. Observe under a microscope and digest for 1-2 min. When the cells are separated and rounded, digestion is complete.

[0045] (3) Quickly discard the trypsin, add DMEM complete medium (for example, a 50-ml centrifuge tube: 5 ml fetal bovine serum, 0.5 ml penicillin-streptomycin mixture, 0.5 ml SCF, 45 ml basal medium), blow the cells to make a single-cell suspension, subculture at a ratio of 1:3, and expand the culture at 37°C and 5% CO2 saturated humidity.

[0046] 4.2 Cell transfection

[0047] (1) Take rat small intestinal cajal interstitial cells in the logarithmic growth phase and in good growth condition, make a single cell suspension with a special culture medium, and set 2×10 per well. 5 The cells were evenly inoculated into 6-well plates and cultured overnight at 37°C and 5% CO2 saturated humidity;

[0048] (2) 2 hours before transfection, change to serum-free medium I;

[0049] (3) Transfection was performed according to the following experimental groups:

[0050] 1) Normal group

[0051] 2) NC group

[0052] 3) MEG3-87

[0053] 4) MEG3-448

[0054] 5) MEG3-1158

[0055] (4) Transfection steps:

[0056] For each transfection sample, prepare as follows:

[0057] a) Dilute 10 μL of the small fragments (concentration 20 μM) with 100 μL serum-free opti-MEM, mix gently with a pipette tip, and let stand at room temperature for 5 minutes;

[0058] b) Gently mix Lipofectamine before use TM 2000, then take 5 μL of Lipofectamine TM 2000 diluted in 100 μL opti-MEM and incubated at room temperature for 5 min;

[0059] c) After standing at room temperature for 5 minutes, mix Lipofectamine TM 2000 and dilution of the small fragment (total volume 200 μL), mix gently and let stand at room temperature for 20 min;

[0060] (5) Add 200 μL of the mixed solution to each culture well and gently shake the cell culture plate back and forth to mix the mixed solution with the culture medium in the culture plate;

[0061] (6) The cells were cultured in a 37°C 5% CO2 incubator. After 6 h, the transfection solution was aspirated and replaced with normal culture medium.

[0062] (7) Continue culturing in a 37°C 5% CO2 incubator for 24 hours before subsequent cell staining and identification.

[0063] 5 Experimental results

[0064] The small fragment sequence is as follows:

[0065]

[0066] After interference:

[0067] MEG3-rat-87 group 0.8×10 5 ng / ml

[0068] MEG3-rat-448 group 0.35×10 5 ng / ml

[0069] MEG3-rat-1158 group 0.6×10 5 ng / ml

[0070] The results showed that compared with the MEG3-87 group and the MEG3-1158 group, the MEG3-448 group had the best interference effect, which was statistically significant.

[0071] First, the MEG3-rat-448 sequence has significant biological activity and can effectively interfere with the function of intestinal Cajal cells. This discovery reveals the potential role of the MEG3 gene in regulating intestinal Cajal cells and provides a new candidate gene fragment for subsequent gene therapy and cell regulation research.

[0072] Secondly, the MEG3-rat-448 sequence has the best interference effect on intestinal Cajal cells, indicating that it may have unique advantages in regulating intestinal function. As an important component of the intestinal nervous system, intestinal Cajal cells play a key regulatory role in intestinal motility, sensory function, and endocrine activity. Therefore, by regulating the function of intestinal Cajal cells, the MEG3-rat-448 sequence is expected to provide new strategies and methods for the treatment of intestinal diseases.

[0073] Finally, this result also proves the effectiveness and reliability of qPCR technology in gene expression analysis and functional screening. Through precise quantitative detection, we can evaluate the interference effects of different gene fragments on specific cell types, providing a strong basis for subsequent gene therapy and cell regulation research.

[0074] In summary, the best result of MEG3-rat-448 interfering with intestinal Cajal cells proved the potential role and value of this fragment in regulating intestinal function, and provided new ideas and methods for subsequent gene therapy and cell regulation research.

[0075] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that are not conceived through creative work should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope defined in the claims.

Claims

1. MEG3-rat-448 sequence, It is characterized in that The primer sequence corresponding to the MEG3-rat-448 sequence is shown in SEQ ID No. 1-2.

2. Use of the MEG3-rat-448 sequence according to claim 1 in inhibiting the function of intestinal Cajal cells.

3. The use according to claim 2, Features: The MEG3-rat-448 sequence regulates intestinal motility, sensory function and endocrine activity by regulating intestinal Cajal cells.

4. Use of the MEG3-rat-448 sequence according to claim 1 as a drug for regulating intestinal function.

5. Use of the MEG3-rat-448 sequence described in claim 1 in virus packaging.

6. The use according to claim 5, Features: The specific operation of the virus packaging of the MEG3-rat-448 sequence is as follows: according to the RNA sequence information of MEG3, PCR amplification primers are designed, and the upstream and downstream primers are respectively added with restriction sites XbaI and EcoRΙ and protective bases, and the upstream primer is added with the kozak sequence. PCR primer sequence: Forward primer-MEG3: 5'-TCTAGA(XbaI)GCCACC(kozak) AGCCCCTAGCGCAGA-3'; Revese primer-MEG3: 5'-GAATTC (Eco RI)TTGTTAAGACAGGAAACAC-3', PCR length is 1592bp. Real Time-PCR detection, primer sequence: Betaactin-forward primer, 5'-CCTGTACGCCAACACAGTGC-3', Beta acin-reverse primer 5'-ATACTCCTGCTTGCTGATCC-3', PCR product length is 211bp; MEG3-forward primer 5'-TGCGGATGGAAGCTGCGGAAGAG-3', MEG3-reverse primer 5'-CGCCCAAACCAGGAAGGAGACGAG-3', PCR product length is 102bp.

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