APOBEC3A gene overexpression cell strain and application thereof in research of bladder cancer
By constructing a stable APOBEC3A overexpressing cell line, we filled the research gap on the impact of APOBEC3A on the malignancy of bladder cancer, achieved stable expression of APOBEC3A in bladder cancer cells, and provided a basis for research tools and therapeutic drug development.
Patent Information
- Application Number
- CN202511159692.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-19
- Publication Date
- 2025-11-07
Smart Images
Figure CN120905312A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of biological medicine, and particularly relates to an APOBEC3A gene overexpression cell strain, a preparation method thereof, and application of the APOBEC3A gene overexpression cell strain in research on influence of APOBEC3A on malignant degree of bladder cancer. BACKGROUND
[0002] APOBEC3A (Apolipoprotein B mRNA-editing enzyme catalytic polypeptide 3A) is a cytidine deaminase in the human body and belongs to the APOBEC3 family. Its core function is to convert cytosine (C) on single-stranded DNA into uracil (U) through deamination, resulting in DNA mutation. Therefore, APOBEC3A is a double-edged sword, which can prevent viruses but excessive activity can trigger cancer-related mutations.
[0003] Bladder cancer is the most common malignant tumor of the urinary system and originates from the urothelial cells of the inner wall of the bladder. Bladder cancer includes luminal, p53-like, basal, and claudin-low molecular subtypes. The expression amount of APOBEC3A is significantly different in different subtypes, which indicates that APOBEC3A may play an important role in the mutation formation of some subtypes of bladder cancer. In addition, APOBEC3A may also be expressed differently in bladder cancers of different malignant degrees.
[0004] However, at present, the influence of APOBEC3A on the malignant degree of bladder cancer has not been deeply studied. Therefore, a stably transfected APOBEC3A overexpression cell strain is needed to study the influence of APOBEC3A on the malignant degree of bladder cancer. SUMMARY
[0005] To solve the above problems, the present application provides a method for constructing a stably transfected APOBEC3A overexpression cell strain, which comprises the step of transferring an overexpression vector containing an APOBEC3A expression cassette into starting cells, wherein the coding gene of APOBEC3A is shown as SEQ ID NO: 1.
[0006] In one specific embodiment, the promoter of the APOBEC3A expression cassette is an EF1A promoter.
[0007] In one specific embodiment, the overexpression vector is a vector based on modification of a lentivirus.
[0008] In one specific embodiment, the overexpression vector contains an anti-puromycin Puro gene as a screening marker.
[0009] In one specific embodiment, the starting cell is mouse bladder cancer cell line MB49 The present application also provides a stably transfected APOBEC3A overexpression cell strain prepared by the above method.
[0010] The present application also provides the use of the above stably transfected APOBEC3A overexpression cell strain in the study of the influence of APOBEC3A on the malignancy of bladder cancer.
[0011] The present application constructs a stably transfected APOBEC3A overexpression cell strain, which stably expresses APOBEC3A during subculture and does not lose APOBEC3A due to cell division. The cell strain can be used to study the influence of APOBEC3A on the malignancy of bladder cancer, and has important significance for the development of prognostic molecular markers and therapeutic drugs for bladder cancer. BRIEF DESCRIPTION OF DRAWINGS
[0012] Figure 1 Map around the insertion sequence of the overexpression vector.
[0013] Figure 2 Map around the insertion sequence of the control vector.
[0014] Figure 3 Microscopic examination of the stably transfected cell strain under a fluorescence microscope.
[0015] Figure 4 Detection of APOBEC3A expression by qPCR. DETAILED DESCRIPTION
[0016] The principles and features of the present application are described below in conjunction with the accompanying drawings, which are only used to explain the present application and are not intended to limit the scope of the present application.
[0017] 1. Construction of expression vector The lentivirus pLV-EF1A-EGFP-2A-Puro plasmid was used to construct the expression vector, and the coding region sequence of human APOBEC3A (APOBEC3A CDS[NM_145699.4], SEQ ID NO:1) was inserted downstream of the promoter EF1A to obtain the APOBEC3A expression vector LV-EF1A>Kozak-Human APOBEC3A CDS[NM_145699.4]-mPGK>EGFP / T2A / Puro, as shown in Figure 1 .
[0018] The control vector is the lentivirus plasmid LV-mPGK>EGFP / T2A / Puro without the coding region of human APOBEC3A, as shown in Figure 2 .
[0019] 2. Construction of stable cell line APOBEC3A expression vector and control vector were respectively transformed into mouse bladder cancer cell line MB49. Transformants were screened by using culture medium containing 4 μg / mL Puro.
[0020] The obtained transformants were subcultured in DMEM culture medium (containing 10% FBS) containing 4 μg / mL Puro. Finally, the stable overexpression cell line (SC756-1) and the control cell line (SC756-2) were obtained respectively.
[0021] 3. Phenotype of stable cell line The cell culture was sampled and observed under a fluorescence microscope. As shown in Figure 3 , the overexpression strain and the control strain both showed green fluorescence, indicating that the plasmid vector was stably present in both cell strains and entered the daughter cells with cell division.
[0022] The expression of APOBEC3A in the cell strain was detected by qPCR, taking mGapdh as an internal standard, and the data was analyzed and processed according to the 2 -ΔΔCT method. The primer sequences used are shown in Table 1.
[0023] Table 1 Primer sequences for qPCR As shown in Figure 4 and Table 2, APOBEC3A was stably expressed in the overexpression group, while no APOBEC3A was found in the control group. It can be seen that APOBEC3A is stably expressed in the stable overexpression cell line constructed by the present application.
[0024] Table 2 qPCR quantitative detection of APOBEC3A The above experiments show that we have constructed a stable APOBEC3A overexpression cell line, and APOBEC3A is stably expressed during subculture of the cell line, and is not lost due to cell division.
[0025] The cell line can be used to study the effect of APOBEC3A on the malignancy of bladder cancer.
[0026] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.
Claims
1. A method of constructing a stable APOBEC3A overexpression cell line, characterized by, The method comprises the step of transferring an overexpression vector containing an APOBEC3A expression cassette into a starting cell, wherein the coding gene of APOBEC3A is shown as SEQ ID NO:
1.
2. The method of claim 1, wherein, The promoter of the APOBEC3A expression cassette is an EF1A promoter.
3. The method of claim 1, wherein, The overexpression vector is a vector based on lentivirus modification.
4. The method of claim 1, wherein, The overexpression vector contains an anti-puromycin Puro gene as a screening marker.
5. The method of claim 1, wherein, The starting cell is a mouse bladder cancer cell line MB49.
6. A stably transfected APOBEC3A overexpressing cell line, characterized in that, Prepared according to any one of claims 1-5.
7. The use of the stable APOBEC3A overexpression cell strain of claim 6 in the study of the influence of APOBEC3A on the malignancy of bladder cancer.