Page 19 - 南京医科大学自然版

第45卷第11期 林书慧，钱萌森，朱 静，等. METTL3介导的KIF11 mRNA m6A修饰通过PI3K/AKT信号通路促进结
2025年11月 直肠癌进展［J］. 南京医科大学学报（自然科学版），2025，45（11）：1546-1562 ·1549 ·

taining 20% FBS to the lower chamber，the cells that whole experiment was operated on ice to maintain
had passed through the membrane were fixed with para⁃ RNA stability.
formaldehyde and stained with crystal violet 36-48 h 1.2.10 RNA stability assays
later. Then，the cells were photographed and observed According to the experimental design，HCT116
to assess cell migration capacity according to previous and DLD1 cells were spread evenly in a 6⁃well plate.
［20］
studies . After 18 h，the 5 μg/mL of actinomycin D was added to
1.2.6 Cell apoptosis assay each well，and the cells were collected after 0，3，and 6 h，
HCT116，DLD1，or SW480 cells were spread respectively. RNA was extracted and subjected to qRT⁃
［22］
evenly in 6 ⁃ well plates，after 48 h，the cells were PCR ，and half⁃life was calculated using the compara⁃
washed twice with pre⁃cooled PBS，followed by resus⁃ tive threshold cycle 2 -ΔΔCt method.
pension of the cells with 1× binding buffer to a concen⁃ 1.3 Statistical analysis
6
tration of 1 × 10 cells/mL；100 μL of cell suspension GraphPad Prism version 9.5 and Image J version
was incubated with 5 μL of Annexin V⁃FITC and 5 μL 5.4 were utilized to analyze the data. Every experiment
of PI staining solution for 15 min in the dark. Flow cy⁃ was independently performed three times. When the
tometry analysis was performed within 1 h. data conformed to normal distribution，the data differ⁃
1.2.7 Subcutaneous xenograft experiments ences between the two groups were examined using the
BALB/c nude mice were administered subcutane⁃ Student’s t⁃test，while ANOVA was used to compare
ous injections of HCT116 cells transfected with shNC/ more than two groups. The Fisher’s exact test was
6
shKIF11⁃1，containing approximately 3.5×10 cells per used to evaluate the clinicopathological data. P < 0.05
150 μL PBS，and the volume and weight of the tumors were consisdered statistically significant.
2
weremeasuredevery four days（volume=length×width /2）.
2 Results
1.2.8 RNA immunoprecipitation（RIP）assay
Following the instructions，adequate amounts of 2.1 KIF11 expression was upregulated in CRC tissues
cells were lyzed with lysates using the Magna RIP kit. According to OmicShare tools（https：//www.omic⁃
［23］
The cells were incubated overnight with anti⁃IGF2BP2 share.com/tools/） and the GEPIA database（http：//ge⁃
or IgG antibody and magnetic beads at 4 ℃. The mag⁃ pia2.cancer ⁃ pku.cn/），the expression of KIF11 was
netic beads were then washed and treated with pro⁃ highly expressed in various types of cancers，including
teinase K to remove proteins. Lastly，the purified RNA colon and rectal cancers（Figure 1A，B）. We examined
was reverse⁃transcribed to cDNA and analyzed using the KIF11 expression levels in tumor tissues and adja⁃
［21］
qRT⁃PCR . cent paired normal tissues from 30 pairs of CRC patients
1.2.9 Methylated RNA immunoprecipitation（MeRIP） by qRT ⁃ PCR. The findings demonstrated that KIF11
The Manga MeRIP m6A kit was used. According was highly expressed in CRC tissues compared with
to the instructions，total cell RNA was extracted and normal tissues（Figure 1C）. Normalized to normal
reduced into 100 or fewer nucleotide fragments. Then tissues，KIF11 expression levels in 30 pairs of the
the magnetic bead⁃coupled anti⁃m6A antibody was CRC tissues were divived into the high and low expres⁃
incubated in immunoprecipitation buffer with the frag⁃ sion groups by qRT⁃PCR（Figure 1D）. Subsequently，
mented RNA for at 4 ℃ 2 h，while an IgG control was we analyzed the relationship between KIF11 expres⁃
set up to exclude nonspecific binding. Next，the magnetic sion and clinicopathological factors in the samples，
bead complexes were washed with pre⁃cooled washing and the results showed that a high level of KIF11 was
buffer to remove nonspecific RNA，and finally the associated with lymph node metastasis and distant
enriched m6A⁃modified RNA was recovered by protein⁃ metastasis（Table 2）.
ase K digestion and phenol⁃chloroform extraction，and 2.2 KIF11 promoted CRC cell proliferation and mi⁃
the resulting products were analyzed by qRT⁃PCR to gration in vitro
determine the methylation level of the target RNA. The CCK⁃8 and colony formation assays revealed that