摘要
目的：探讨驱动蛋白家族成员11（kinesin family member 11，KIF11）在结直肠癌（colorectal cancer，CRC）中的生物学功能及其分子调控机制。方法：采用定量实时PCR技术结合公共数据库分析KIF11在CRC中的表达情况。通过CCK-8细胞增殖实验、集落形成实验、EdU 染色实验和 Transwell 迁移实验评估 KIF11 对 CRC 细胞增殖和迁移能力的影响。运用 Western blot、RNA免疫共沉淀定量PCR（RIP-qPCR）、甲基化RNA免疫共沉淀定量PCR（MeRIP-qPCR）和RNA稳定性实验解析KIF11的 N6-甲基腺苷（N6-methyladenosine，m6A）修饰机制。采用RNA测序技术和相关性分析方法探究KIF11调控的下游机制。结果：KIF11在CRC组织中高表达，其表达水平与CRC细胞的增殖和迁移能力呈正相关。甲基转移酶样蛋白3（methyltransferase- like 3，METTL3）和胰岛素样生长因子 2 mRNA 结合蛋白 2（insulin like growth factor 2 mRNA binding protein 2，IGF2BP2）通过 m6A修饰调控KIF11 mRNA的稳定性，从而正向调控KIF11的表达。进一步研究发现，KIF11通过激活PROM1/PI3K/AKT通路促进CRC的恶性进展。结论：METTL3/IGF2BP2介导的KIF11 mRNA m6A修饰通过PI3K/AKT信号通路促进CRC的发生发展，提示KIF11可能作为CRC潜在的预后标志物和治疗靶点。
关键词
Abstract
Objective：To investigate the biological functions and molecular regulatory mechanisms of kinesin family member 11 （KIF11）in colorectal cancer（CRC）. Methods：The expression of KIF11 in CRC was examined by qRT-PCR and public databases. Functional assays（CCK - 8，colony formation，EdU，and Transwell）were employed to evaluate KIF11’s roles in CRC progression. Western blot，RIP-qPCR，MeRIP-qPCR，and RNA stability assays were performed to elucidate the molecular mechanism of N6 - methyladenosine（m6A）modification for KIF11. RNA sequencing（RNA-seq）and correlation analysis were used to examine the downstream mechanism of KIF11 regulation. Results：KIF11 was highly expressed in CRC and promoted CRC proliferation and migration. Mechanistically，methyltransferase-like 3（METTL3）/insulin like growth factor 2 mRNA binding protein 2（IGF2BP2） enhanced KIF11 mRNA stability and expression in an m6A-dependent way. Furthermore，by means of the PROM1/PI3K/AKT pathway， KIF11 facilitated the progression of CRC. Conclusion：The m6A modification of KIF11 by METTL3/IGF2BP2 contributes to CRC progression via the PI3K/AKT signaling pathway，highlighting its potential as a prognostic biomarker and therapeutic target.
Keywords
Colorectal cancer（CRC）is the third most com⁃ mon cancer globally and the second leading cause of death from cancer[1] . According to GLOBOCAN 2020 estimates，3 154 674 new CRC cases are projected worldwide by 2040[2]，highlighling its significance as a public health crisis. Despite advances in diagnostic and therapeutic strategies，patient prognosis remains poor[3] . Thus，exploring the molecular mechanisms of CRC and promising therapeutic targets are essential.
Kinesin family member 11（KIF11），a plus ⁃end ⁃ directed kinesin motor protein of the kinesin family， plays a critical role in spindle pole separation and chro⁃ mosome alignment during mitosis[4] . Accumulating evi⁃ dence denonstrates that KIF11 upregulation is associat⁃ ed with poor prognosis in many cancers. For example， KIF11 is highly expressed and promotes cell prolifera⁃ tion in gallbladder cancer[5]；in hepatocellular carcino⁃ ma，KIF11 is negatively correlated with senescence biomarkers；its knockdown induces cellular senes⁃ cence and suppresses hepatocellular carcinoma（HCC） progression[6] . Epigenetic modifications，particularly N6 ⁃ methyladenosine（m6A），are pivotal in disease pathogenesis. As the most prevalent internal mRNA modification in humans（affecting almost 90% of transcripts）[7]，m6A regulates post⁃transcriptional pro⁃ cesses. However，whether m6A modifications and their regulatory enzymes control KIF11 expression in CRC remains unexplored.
The dynamic regulation of m6A methylation modi⁃ fication is performed by methyltransferases（“writers”）， demethylases（“erasers”），and binding proteins（“read⁃ ers”）[8] . METTL3 is one of the major methyltransferases proven to be highly expressed in various cancers and to accelerate tumor malignant development，including colorectal and lung cancers[9-12] . Similarly，IGF2BP2 is a key m6A modification“reader”that encourages the growth of malignancies by affecting the stability and translation of downstream target mRNAs[13-15].
PROM1，also known as CD133，is a cancer stem cell（CSC）marker involved in tumorigenesis，drug re⁃ sistance，and apoptosis in CSCs. For example，CD133 can promote angiogenesis through VEGF ⁃A[16]；it can also promote gastric cancer（GC）progression through the PI3K/AKT signaling pathway[17].
In this study，we demonstrated that KIF11 was highly expressed in CRC，and its knockdown inhibited the proliferation and migration of CRC cells. We also found that the METTL3 ⁃ IGF2BP2 axis regulated KIF11 in an m6A ⁃ dependent way. In addition，KIF11 promoted CRC progression by regulating PROM1 and PI3K/AKT signaling pathway. Collectively，these find⁃ ings position KIF11 as a potential therapeutic target and prognostic biomarker for CRC.
1 Materials and Methods
1.1 Materials
1.1.1 Clinical samples
This study collected 30 pairs of CRC tissues and adjacent normal tissues from patients undergoing sur⁃ gery at the Second Affiliated Hospital of Nanjing Medi⁃ cal University. Before surgery，none of the patients underwent radiotherapy or chemotherapy. All patients who participated in this study signed informed consent forms. The whole process was in strict accordance with the Declaration of Helsinki. The ethical approval of this study was provided by the Institutional Review Board of Nanjing Medical University（Ethics No.2024⁃ KY⁃274⁃01）.
1.1.2 Animals
Four ⁃ week ⁃ old，16-18 g specific pathogen free （SPF）male BALB/c nude mice were purchased from GemPharmatech Co.，Ltd. The nude mice were housed in an environment of 20-27℃ with relative humidity of 40%-60%. The Nanjing Medical University Animal Ethics Committee granted its approval for animal stud⁃ ies（IACUC⁃2410071）.
1.1.3 Reagents and instruments
TRIzol（Invitrogen，USA）；anti ⁃ KIF11，anti ⁃ IGF2BP2，anti ⁃ ACTIN，anti ⁃ GAPDH（Proteintech， USA）；anti ⁃METTL3（Youmengbiology，China）；anti ⁃ PI3K，anti⁃p⁃PI3K，anti⁃AKT（CST，USA）；p⁃AKT（Af⁃ finity，USA）；anti⁃IgG（Millipore，USA）；SYBR Green premix（CWBIO，Taizhou，China）；BeyoClickTM EdU cell proliferation kit with AF555（Beyotime，Shanghai， China）；Annexin V ⁃ FITC/PI Apoptosis Detection Kit （Vazyme，Nanjing，China）；Magna RIP kit，anti⁃m6A， IgG antibody（Millipore，USA）. Light Cycler480Ⅱreal⁃ time fluorescence PCR Instrument（Roche，USA）；Cen⁃trifuges（Eppendorf，Germany）；Cell incubator（Ther⁃ moFisher Scientific，USA）；Chemiluminescence imager （Tanon，Guangdong，China）；Electrophoresis appara⁃ tus，Thermal cycler（BIORAD，USA）；Flow cytometer （Beckman Coulter，USA）；Fluorescence microscope （Olympus，Japan）；Microplate reader（Biotek，USA）.
1.2 Methods
1.2.1 Lentiviral and plasmid transfections
shRNA and overexpression plasmids were de⁃ signed for genes including KIF11. They were transfect⁃ ed into cells according to the instructions[18] .
1.2.2 RNA Extraction and qRT⁃PCR
Total cellular RNA was extracted. The cells were fully lyzed using TRIzol reagent and allowed to stand for 5 min. Then 0.2 mL of chloroform was added and the mixture was shaken vigorously for 15 s，followed by a3⁃minute rest，The sample was centrifuged at 12 000 g at 4℃ for 15 min. We transfered the upper layer of liquid to a new centrifuge tube，added 0.5 mL of isopro⁃ panol to precipitate the RNA follaued by a10⁃minute rest，and then centrifuged the sample at 12 000 g at 4℃ for 10 min. Subsequently，we discarded the supernatant and then washed the RNA precipitate with 75% ethanol twice，and finally dissolved the RNA precipitate with 20-50 μL of RNase ⁃free water. SYBR Green premix was used for qRT ⁃ PCR. The comparative threshold cycle 2-ΔΔCT method was used to evaluate the relative gene expression and normalize it to GAPDH[19] . The primer sequences used for qRT ⁃ PCR are shown in Table1.
表1引物序列表
Table1Primer sequences
1.2.3 Western blot
Total protein of cells was extracted and then sepa⁃ rated by 10% SDS ⁃ PAGE. The concentrated gel was 80 V and the separated gel was 120 V for constant voltage electrophoresis and then transferred the protein to a PVDF membrane. After sealing the membrane with 5% BSA for 2 h，the primary antibody was incu⁃ bated overnight at 4℃ . The membrane was rinsed in TBST for 10 min on day 2 and repeated three times， followed by incubation with the corresponding second⁃ ary antibody for 2 h. Finally，an ECL chemilumines⁃ cent solution was used to visualize the protein bands.
1.2.4 CCK⁃8 assay，colony formation，and EdU exper⁃ iments
To determine cell proliferation，a CCK⁃8 test was performed. According to the experimental design， HCT116，DLD1，or SW480 cells were spread in 96 ⁃ well plates at a density of 2 000 cells per well. After 24-36 h，CCK ⁃ 8 solution was added for another 24， 36，48，72，and 96 h. The absorbance at 450 nm was detected after 2 h of incubation. Cells were spread at a density of 1 000-2 000 cells per well for colony for⁃ mation experiments in 6⁃well plates，and the cell colo⁃ nies were fixed with paraformaldehyde after 10-14 days and then stained with crystal violet. For EdU experi⁃ ments，HCT116，DLD1，and SW480 cells were spread evenly in 96⁃well plates at a density of 5 000 cells per well. After 24 h，the96 ⁃ well plates were incubated with 20 μmol/L EdU working solution for 2 h. Subse⁃ quently，the medium was discarded，and the cells were washed three times with PBS. Then 4% paraformalde⁃ hyde was added and fixed at room temperature for 15 min，followed by permeabilization with 0.5% Triton X⁃100 for 10 min to enhance cell membrane permeabil⁃ ity. Next，the cells were incubated with click reaction buffer for 30 min away from light. and the cell prolifera⁃ tion ratio was detected under a fluorescence microscope.
1.2.5 Transwell experiments
After adding 60 000-80 000 HCT116，DLD1，or SW480 cells to the upper chamber and medium con⁃taining 20% FBS to the lower chamber，the cells that had passed through the membrane were fixed with para⁃ formaldehyde and stained with crystal violet 36-48 h later. Then，the cells were photographed and observed to assess cell migration capacity according to previous studies[20].
1.2.6 Cell apoptosis assay
HCT116，DLD1，or SW480 cells were spread evenly in 6 ⁃ well plates，after 48 h，the cells were washed twice with pre ⁃cooled PBS，followed by resus⁃ pension of the cells with 1× binding buffer to a concen⁃ tration of 1 × 106 cells/mL；100 μL of cell suspension was incubated with 5 μL of Annexin V⁃FITC and 5 μL of PI staining solution for 15 min in the dark. Flow cy⁃ tometry analysis was performed within 1 h.
1.2.7 Subcutaneous xenograft experiments
BALB/c nude mice were administered subcutane⁃ ous injections of HCT116 cells transfected with shNC/ shKIF11⁃1，containing approximately 3.5×106 cells per 150 μL PBS，and the volume and weight of the tumors weremeasuredevery four day（s volume=length×width2 /2）.
1.2.8 RNA immunoprecipitation（RIP）assay
Following the instructions，adequate amounts of cells were lyzed with lysates using the Magna RIP kit. The cells were incubated overnight with anti⁃IGF2BP2 or IgG antibody and magnetic beads at 4℃. The mag⁃ netic beads were then washed and treated with pro⁃ teinase K to remove proteins. Lastly，the purified RNA was reverse ⁃transcribed to cDNA and analyzed using qRT⁃PCR[21].
1.2.9 Methylated RNA immunoprecipitation（MeRIP）
The Manga MeRIP m6A kit was used. According to the instructions，total cell RNA was extracted and reduced into 100 or fewer nucleotide fragments. Then the magnetic bead ⁃ coupled anti ⁃ m6A antibody was incubated in immunoprecipitation buffer with the frag⁃ mented RNA for at 4℃ 2 h，while an IgG control was set up to exclude nonspecific binding. Next，the magnetic bead complexes were washed with pre ⁃cooled washing buffer to remove nonspecific RNA，and finally the enriched m6A⁃modified RNA was recovered by protein⁃ ase K digestion and phenol ⁃chloroform extraction，and the resulting products were analyzed by qRT ⁃ PCR to determine the methylation level of the target RNA. The whole experiment was operated on ice to maintain RNA stability.
1.2.10 RNA stability assays
According to the experimental design，HCT116 and DLD1 cells were spread evenly in a6⁃well plate. After 18 h，the5 μg/mL of actinomycin D was added to each well，and the cells were collected after 0，3，and 6 h， respectively. RNA was extracted and subjected to qRT⁃ PCR[22]，and half⁃life was calculated using the compara⁃ tive threshold cycle 2-ΔΔCt method.
1.3 Statistical analysis
GraphPad Prism version 9.5 and Image J version 5.4 were utilized to analyze the data. Every experiment was independently performed three times. When the data conformed to normal distribution，the data differ⁃ ences between the two groups were examined using the Student’s t⁃test，while ANOVA was used to compare more than two groups. The Fisher’s exact test was used to evaluate the clinicopathological data. P <0.05 were consisdered statistically significant.
2 Results
2.1 KIF11 expression was upregulated in CRC tissues
According to OmicShare tools（https：//www.omic⁃ share.com/tools/）[23] and the GEPIA database（http：//ge⁃ pia2.cancer ⁃ pku.cn/），the expression of KIF11 was highly expressed in various types of cancers，including colon and rectal cancers（Figure1A，B）. We examined the KIF11 expression levels in tumor tissues and adja⁃ cent paired normal tissues from 30 pairs of CRC patients by qRT ⁃ PCR. The findings demonstrated that KIF11 was highly expressed in CRC tissues compared with normal tissues（Figure1C）. Normalized to normal tissues，KIF11 expression levels in 30 pairs of the CRC tissues were divived into the high and low expres⁃ sion groups by qRT ⁃ PCR（Figure1D）. Subsequently， we analyzed the relationship between KIF11 expres⁃ sion and clinicopathological factors in the samples， and the results showed that a high level of KIF11 was associated with lymph node metastasis and distant metastasis（Table2）.
2.2 KIF11 promoted CRC cell proliferation and mi⁃ gration in vitro
CCK ⁃8 and colony formation assays revealed that the knockdown of KIF11 inhibited the proliferative ability of cells（Figure 2A，B，D，E），while overex⁃ pression of KIF11 enhanced cell viability（Figure 2C， F，G）. In addition，the EdU assay revealed the same re⁃ sults（Figure 2H-K）. According to transwell assay， KIF11 knockdown weakened the migration ability of cells compared with the control，while overexpression of KIF11 significantly enhanced the cell migration capacity（Figure 2L-O）. Flow cytometry analysis revealed that KIF11 knockdown significantly increased apoptosis rate in CRC cells，whereas KIF11 overexpres⁃ sion suppressed cell apoptosis（Figure3）.
图1KIF11在结直肠癌组织中高表达
Figure1KIF11 was highly expressed in CRC tissues
表2CRC患者KIF11的表达水平与临床病理参数的相关性
Table2Correlations between KIF11 expression and clini⁃ copathological characteristics in CRC patients
2.3 KIF11 promoted CRC progression in vivo
To explore whether KIF11 promotes the progres⁃ sion of CRC in vivo，we conducted subcutaneous xeno⁃ graft experiments. HCT116 cells transfected with shNC or shKIF11⁃1 were inoculated into mice（five mice per group），and the tumors were compared after 21 days of injection（Figure4A）. The results showed that com⁃ pared with the control group，the subcutaneous tumors with KIF11 knockdown exhibited lower volume and weight（Figure4B，C）.
2.4 METTL3 ⁃ regulated m6A modification enhanced KIF11 stability through IGF2BP2 ⁃ dependent mecha⁃ nisms
图2KIF11促进结直肠癌进展
Figure2KIF11 promoted colorectal cancer cell progression
图3KIF11表达影响CRC细胞凋亡
Figure3KIF11 expression affected apoptosis in CRC cells
To explore potential mechanisms regulating KIF11 expression，we employed the starBase database （https：//rnasysu.com/encori/）and the STRING website （https：//cn.string ⁃ db.org/）to conduct an enrichment analysis of RNA binding proteins（RBPs）connected with KIF11 mRNA. The results demonstrated a strong correlation between m6A modification and KIF11 mRNA（Figure5A）. Subsequently，we use the SRAMP website（http：//www.cuilab.cn/sramp/）to confirm the existence of several potentially high ⁃ confidence m6A modification sites in the KIF11 mRNA（Figure5B）. The online prediction site（http：//m6a2target.cancer⁃ omics.org）revealed that METTL3 mediated the m6A modification of KIF11. According to previous research， METTL3 was highly expressed in CRC and associated with poor prognosis [10] . GEPIA database showed that METTL3 was positively correlated with KIF11 expres⁃ sion（Figure5C）. We verified that METTL3 positively regulated KIF11 by qRT ⁃ PCR and WB experiments （Figure5D-G）. To verify whether KIF11 mRNA was subject to METTL3 ⁃ mediated m6A modification，we performed MeRIP⁃qPCR，and the results revealed that the m6A abundance of KIF11 mRNA was significantly reduced after METTL3 knockdown（Figure6A）. The above results suggest that METTL3 mediates the m6A methylation modification of KIF11. Furthermore，we found that KIF11 mRNA stability was decreased after the knockdown of METTL3（Figure6B，C）. Overall， METTL3 induced KIF11 m6A modification and stabi⁃ lized its expression. The current consensus indicates that the m6A modifications function primarily by re⁃ cruiting“reader”proteins. Earlier research has report⁃ed that the IGF2BPs family is a crucial“reader”of m6A modification，which affects gene expression by regulating the stability of target mRNAs[24] . Therefore， we used the starBase database to predict the relation⁃ ship between KIF11 and their expression，and found that the correlation between IGF2BP2 and KIF11 was the highest（Supplementary Figure2）. The RIP ⁃qPCR experiment results demonstrated that IGF2BP2 and KIF11 interacted with each other（Figure6D）. Accord⁃ ing to qRT ⁃ PCR and WB assay results，IGF2BP2 knockdown decreased the mRNA and protein levels of KIF11（Figure6E-H）. In addition，the RNA stability assay demonstrated that KIF11 mRNA stability de⁃ creased after IGF2BP2 knockdown（Figure6I，J）. These findings indicated that IGF2BP2 improved mRNA stability and functions as a“reader”of KIF11 mRNA methylation. Rescue experiments revealed that METTL3 overexpression partially counteracted the re⁃ duction of KIF11 expression levels caused by IGF2BP2 silencing（Figure6K）. Taken together，MET⁃ TL3 ⁃ mediated m6A modification enhances KIF11 mRNA stability in an IGF2BP2⁃dependent manner.
图4KIF11在体内促进CRC进展
Figure4KIF11 promoted CRC progression in vivo
2.5 METTL3 promoted CRC proliferation and migra⁃ tion by regulating KIF11
Based on these results，we further investigated the impact and regulatory mechanisms of METTL3 on the proliferation and migration of CRC cells. The results of CCK⁃8（Figure7A，B），colony formation（Figure7C，D）， and EdU（Figure7E，F）experiments showed that MET⁃ TL3 silencing inhibited the proliferative ability of CRC cells，while overexpression of KIF11 partially rescued the adverse effect. In addition，Transwell assay results demonstrated that KIF11 overexpression partially elim⁃ inated the inhibitory effect on migration caused by METTL3 knockdown in CRC cells（Figure7G，H）. Consequently，KIF11 was discovered to be a down⁃ stream target of METTL3 to facilitate CRC progression.
2.6 KIF11 promoted CRC progression through the PI3K/AKT signaling pathway
To further explore the possible downstream molec⁃ ular mechanisms of KIF11，we performed transcrip⁃ tome sequencing analysis. We found a total of 166 dif⁃ ferentially expressed genes（DEGs）between the knock⁃ down and control groups，comprising114 upregulated genes and 52 downregulated genes in KIF11 knock⁃ down cells（P <0.05，|log2（fold change）| >1）（Figure8A，B）. By gene set enrichment analysis（GSEA），we found that KIF11 and PI3K/AKT signaling pathway were closely related（Figure8C）. WB results indicated that the knockdown of KIF11 led to significantly lower phosphorylation levels of PI3K and AKT than that of the NC group，whereas the total protein levels of PI3K and AKT were unaffected（Figure8D）. In addition， we assessed the impact of the METTL3⁃KIF11 axis on the PI3K/AKT pathway. We overexpressed KIF11 in METTL3 ⁃ silenced colorectal cancer cells and found that upregulation of KIF11 partially recovered the phosphorylation level of the PI3K/AKT signaling pathway（Figure8E）.
Among the top 10 down ⁃ regulated downstream genes，PROM1 attracted our attention. Previously， Wei et al[25]reported that CD133⁃p85 interaction acti⁃ vated the PI3K/Akt pathway to promote tumorigenicity of glioma stem cells；Zhu et al[26] found that CD133 pro⁃ moted the chemoresistance to 5 ⁃ fluorouracil in GC cells by activating the PI3K/Akt/p70S6K pathway. So we hypothesized that KIF11 might promote CRC pro⁃ gression through the PROM1/PI3K/AKT pathway. The results of qRT⁃PCR and WB demonstrated that PROM1 expression decreased after KIF11 was knocked down， while overexpression of KIF11 produced the opposite effect（Figure9A-D）. CCK ⁃ 8，colony formation，andEdU assays showed that KIF11 knockdown had reduced cell proliferation ability，which could be rescued by PROM1 overexpression（Figure9E-J）. Transwell assay demonstrated that overexpression of PROM1 partially rescued the decreased cell migration ability caused by KIF11 knockdown（Figure10A，B）. The WB results revealed that silencing of KIF11 suppressed the activa⁃ tion of the PI3K/AKT pathway，and the overexpression of PROM1 rescued the KIF11 knockdown⁃mediated in⁃ hibition（Figure10C）. These results suggest that KIF11 promotes CRC progression through the PROM1/ PI3K/AKT pathway.
图5METTL3对KIF11具有正向调控作用
Figure5METTL3 positively regulated KIF11 exptression
图6METTL3介导的m6A修饰通过IGF2BP2依赖性机制增强KIF11的稳定性
Figure6METTL3⁃regulated m6A modification enhanced KIF11 stability through IGF2BP2⁃dependent mechanisms
3 Discussion
Due to tumor recurrence，metastasis and drug re⁃ sistance development，the5⁃year survival rate of CRC is less than 40%[27] . The increasing morbidity and mor⁃ tality suggest that the exploration of novel biomarkers is necessary for the diagnosis and prognosis of colorec⁃ tal cancer. KIF11 is a member of the kinesin family of proteins，which plays a vital role in the mitotic pro⁃ cess[28-29] . Kinesin family proteins（KIFs）are a group of conserved microtubule ⁃ dependent molecular motor proteins that possess ATPase activity and motility prop⁃ erties and are mainly involved in a series of intracellular activities，including cytokinesis，intracellular vesicle and organelle trafficking，and microtubule cytoskeleton reorganization[30-31] . Studies have shown that the kine⁃ sin family is essential to cancer development. For ex⁃ ample，KIF20A regulates malignant proliferation and chemotherapy tolerance in CRC through the JAK2/ STAT3 signaling pathway[32]；KIF11 promotes the pro⁃ gression of hepatocellular carcinoma via the OCT4 pathway[33] . Our study demonstrated that KIF11 was highly expressed in CRC and promoted CRC prolifera⁃ tion and migration.
图7METTL3通过调控KIF11促进CRC增殖和迁移
Figure7METTL3 promoted CRC proliferation and migration by regulating KIF11
图8KIF11通过PI3K/AKT信号通路促进CRC进展
Figure8KIF11 promoted CRC progression through the PI3K/AKT signaling pathway
图9KIF11通过调控PROM1促进CRC增殖
Figure9KIF11 promoted CRC proliferation by regulating PROM1
图10KIF11通过调控PROM1促进CRC迁移
Figure10KIF11 promoted CRC migration by regulating PROM1
Studies have shown that KIF11 up⁃regulation can lead to centrosome fragmentation and chromosome in⁃ stability through multiple signaling pathways，which leads to exuberant cell division and proliferation，and promotes the progression of tumors，including lung and gastric cancers，and glioblastoma[34-35] . To explore the molecular mechanisms by which KIF11 regulates CRC development，we utilized databases and found that KIF11 was closely associated with m6A methylation modifications. RNA methylation modification is a kind of modification that widely exists in all types of RNAs， and its leading role is to control the target protein ex⁃ pression by regulating the transcription，stability，and translation efficiency of mRNA. They can regulate the activity of signaling pathways by acting directly or indi⁃ rectly on key molecules[36-37] . It was demonstrated that METTL3 mediated the m6A modification of KIF11 and affected its mRNA stability. Moreover，rescue experi⁃ ments showed that the overexpression of KIF11 partial⁃ ly restored the tumor suppressor phenotype induced by the knockdown of METTL3. METTL3 is one of the ma⁃ jor“writer”proteins，which has been shown to have a dual function in promoting or suppressing cancers. For example，He et al[38] showed that METTL3 was lowly ex⁃ pressed in PTC and acted as a suppressor in regulating neutrophil infiltration；many studies have also indicat⁃ ed that METTL3 is highly expressed in CRC tissues and is strongly associated with poor prognosis. For ex⁃ ample，METTL3 promotes CRC development by regu⁃ lating the m6A ⁃CRB3⁃Hippo axis[39] . METTL3 inhibi⁃ tion suppresses cell growth and survival in CRC via ASNS downregulation[40]；It has also been found that METTL3 promotes cellular senescence of CRC via modulation of CDKN2B transcription and mRNA sta⁃ bility[41] . Yang et al[42] reported that METTL3⁃mediated RanGAP1 recruited YTHDF1 to facilitate the develop⁃ ment of CRC via the MAPK pathway. Similarly， IGF2BP2 promotes cancer progression by influencing the stability of downstream targets and post ⁃transcrip⁃ tional regulation. Our study，for the first time，proved that the m6A methylation of KIF11 mediated by MET⁃ TL3 was recognized by IGF2BP2，which in turn impact⁃ ed its expression. Subsequently，through RNA ⁃ seq analysis，we found that KIF11 promoted CRC progres⁃ sion by enhancing phosphorylation levels of the PI3K/ AKT pathway. Previous studies have shown that KIF11 also enhances the chemosensitivity of CRC to oxaliplat⁃ in through p53/GSK3β signaling[43] . Aberrant activa⁃ tion of the PI3K/AKT pathway regulates autophagy and EMT levels，induces tumor cell proliferation and migra⁃ tion，and also allows tumor cells to evade apoptosis by creating a dysregulation between anti⁃apoptotic and pro ⁃ apoptotic genes [44] . Our study demonstrated that METTL3 silencing considerably attenuated the phos⁃ phorylation levels of PI3K and AKT，while overexpres⁃ sion of KIF11 in METTL3 ⁃ knockdown cells partially recovered their phosphorylation levels. These results support the idea that METTL3/IGF2BP2 promotes CRC progression through KIF11 by activating the PI3K/AKT signaling pathway.
Our study also revealed that PROM1 was a down⁃ stream target of KIF11. Overexpression of PROM1 in KIF11 ⁃ knockdown cells could rescue the inhibitory effect of KIF11 on CRC proliferation and migration. In addition，KIF11 was able to promote CRC progression through the PROM1/PI3K/AKT pathway.
Despite these conclusions drawn from our study， several limitations remain. First，while we have estab⁃ lished that METTL3 mediates the m6A methylation modification of KIF11，we have not experimentally identified the specific m6A modification sites. Second， although we have recognized the PROM1/PI3K/AKT pathway as a downstream target of KIF11，the precise regulatory mechanisms involved remain to be elucidat⁃ ed. Furthermore，whether KIF11 also promotes tumori⁃ genesis in vivo has yet to be confirmed. In addition，we acknowledge as a limitation that multiple testing cor⁃ rections such as the Bonferroni or Benjamini⁃Hochberg procedures were not applied in the current analysis. In our future research，we will expand the sample size and use more refined correction strategies to enhance sta⁃ tistical power and further improve the reliability of our research conclusions. Therefore，we will continue our experiments to refine this investigation.
4 Conclusion
In conclusion，our study elucidates a novel mecha⁃ nism through which KIF11 regulates colorectal cancer. We demonstrate that METTL3/IGF2BP2 modulates KIF11 expression in an m6A⁃dependent manner，influ⁃ encing its stability and thereby regulating PROM1 and activating the PI3K/AKT signaling pathway to facili⁃ tate CRC progression. These findings underscore the potential of KIF11 as a promising predictive biomarker and an effective therapeutic target，offering new in⁃ sights into the diagnosis and treatment of CRC.
利益冲突声明：
所有作者声明无利益冲突。
Conflicts of Interests：
The authors declare no conflict of interests.
作者贡献声明：
林书慧完成实验验证和初稿撰写；朱静负责研究构思和实验设计；钱萌森和丁洁完成数据整理与分析；丁洁和李娟负责项目资金获取；罗茜和李杰完成数据收集；王科明和王娟完成论文的修改与审阅。
Author’s Contributions：
LIN Shuhui completed the experimental verification and initial draft writing；ZHU Jing was responsible for research con⁃ ception and experimental design；QIAN Mengsen and DING Jie performed the data organization and analysis；DING Jie and LI Juan managed the project funding acquisition；LUO Qian and LI Jie carried out the data collection；WANG Keming and WANG Juan completed the paper revision and review.
