Enhanced Expression of MIC1 Protein in Mouse Forestomach Carcinoma Cells Suppresses Cell Growth and Promotes Apoptosis

Materials and Methods

2.1 Cell Culture and Treatment

The MFC cell line was procured from Wuhan Procell Life Technology Co., Ltd. and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and antibiotics at 37°C in a humidified incubator containing 5% CO2. Cells were seeded at a density of 1×106 cells/well in a 6-well plate. Transfection was conducted using Lipofectamine TM 2000 according to the manufacturer's protocol. After a 24-hour incubation period, cells were divided into control and pEGFP-N1-MIC1 groups. The transfection procedure involved separate dissolution of the recombinant plasmid pEGFP-N1-MC1 and Lipofectamine TM 2000 in serum-free RPMI-1640 culture medium, followed by mixing and incubation. The resulting plasmid-lipid mixture was then added to the 6-well plate and incubated at 37°C with 5% CO2 for 6 hours. Subsequently, the medium was replaced with fresh culture medium containing serum without antibiotics, and cells were further cultured for an additional 24 hours. Cell growth and enhanced green fluorescent protein (EGFP) expression were monitored using a fluorescence microscope.

2.2 Extraction and Reverse Transcription of Mouse Gastric Tissue RNA and PCR Amplification of MIC-1 Gene Sequence

Healthy mouse gastric tissue was harvested and total RNA was extracted using Trizol reagent. RNA integrity was assessed using a 1% agarose gel electrophoresis. Intact RNA was then reverse transcribed into cDNA using 5× Prinescript RT Master Mix. The MIC-1 gene sequence was obtained from the Gene Bank database, and primers for MIC-1 were designed with HindIII and EcoRI restriction endonuclease sites incorporated into the forward and reverse primers, respectively. The PCR reaction mixture consisted of GoTaq qPCR Master Mix, forward and reverse primers, MIC-1 DNA, and sterile water. The resulting PCR products were subsequently analyzed by agarose gel electrophoresis.

2.3 Construction and Detection of Recombinant Plasmid pMD18T-MIC1

PCR products, pMD18T vector, and ligation solution were combined for ligation and subsequently transformed into competent E. coli DH5α. Plasmid extraction was performed on the resulting bacterial culture, and QuickCut HindIII and QuickCut EcoRI restriction enzymes were employed for plasmid digestion. The digestion products were analyzed by agarose gel electrophoresis, and the plasmid with the correct restriction patterns was sent for sequencing analysis.

2.4 Construction and Detection of pEGFP-N1-MIC1 Plasmid

Fragments of pMD18T-MIC1 plasmid with confirmed correct sequencing were excised from the agarose gel using QuickCut HindIII and QuickCut EcoRI enzymes and then recovered using a gel recovery kit. Large fragments of pEGFP-N1 plasmid were digested with the same enzymes, and the recovered fragments were utilized for ligation. The ligation product was subjected to sequencing analysis to verify the correctness of the final recombinant plasmid, designated as pEGFP-N1-MIC1.

2.5 Cell Transfection

MFC cells were seeded in a 6-well plate and transfected with the pEGFP-N1-MIC1 plasmid using Lipofectamine TM 2000. After a 6-hour incubation, the medium was replaced, and cells were further cultured for an additional 24 hours. Transfection efficiency was evaluated by observing cell growth and enhanced green fluorescent protein (EGFP) expression under a fluorescence microscope.

2.6 Western Blotting to Identify MIC1 Protein Expression

Total protein was extracted from transfected cells and subjected to Western blot analysis using an anti-FLAG-Tag antibody. The expression of MIC1 protein was detected using chemiluminescence.

2.7 Cell Cycle Analysis

Cells were harvested 48 hours post-transfection and fixed with ethanol. Cell cycle analysis was performed using flow cytometry.

2.8 Cell Migration Assay

A scratch assay was conducted to assess cell migration. Culture inserts were placed in a six-well plate, and images were captured 24 hours later to analyze the experimental outcomes.

2.9 Colony Formation Assay

Transfected cells were seeded in six-well plates at low density, and clonal colonies were allowed to form over a 12-day period. The number of clonal colonies containing at least 40 cells was quantified.

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