N6-methyladenosine of TRIM27 enhances the stem cell-type phenotype of cisplatin-resistant colorectal cancer cells

Colorectal cancer (CRC), classified as a lethal form of cancer, substantially threatens human well-being. Cancer stem cells (CSCs) reflect subsets for cancerous cells having basic stem-cell type properties, being significantly involved in the development of chemoresistance and tumor relapsing. The aberrant TRIM27 expression in various types of cancer indicates its potential involvement in cancer growth and progression. The current understanding of the TRIM27 involvement in CRC remains limited. In current study indicated that TRIM27 can potentially promote CSC-type phenotype of Cisplatin (DDP)-resistant CRC cells. YTHDF1 recruitment onto m6A-amended TRIM27 was crucial for facilitating the TRIM27 translating process in DDP-resistant CRC cells. The present research proposes that TRIM27 exhibits an oncogenic role by enhancing the CSC-type properties in DDP-resistant CRC via the m6A-modified pathway. The potential therapy for combating the relapse of CRC may include TRIM27 and YTHDF1, as they have been found to have significant roles in promoting CSC-type phenotypic characteristics.


Introduction
Colorectal cancer (CRC) is a prevalent malignancy responsible for significant cancer-related deaths globally, with an elevated mortality rate and adverse prognosis in cancer patients [1].Cisplatin, an antineoplastic agent derived from platinum, has extensively demonstrated its efficacy in treating various malignancies [2].The clinical use of cisplatin results in notable improvements in the prognosis and survival rates of cancer patients.However, its widespread implementation is hindered by substantial challenges, including the emergence of drug resistance and the occurrence of significant side effects [2].The evidence consistently demonstrates that CRC exhibits a notable heterogeneity and covers a small fraction of tumor-inducing cells called cancer stem cells (CSCs).They perform a crucial function in replenishing chemotherapy-resistant CRC, thereby facilitating continuous growth and relapse [3].Despite the effectiveness of conventional therapies in eliminating primary tumors, the presence of CSCs within the malignant tissue can contribute to recurrence due to their inherent CSC characteristics, including self-renewal, differentiation, and tumorigenesis.Exploration of tumor properties resembling CSCs can be effectively conducted via induction of subcutaneous tumor development in BALB/c nude mice [4].Hence, targeting CSCs presents significant therapeutic prospects in addressing the advancement and relapse of CRC.Therefore, comprehensive cellular attributes while self-regenerating capacity by CRC CSCs offers valuable opportunities for developing treatment strategies for CRC.
The tripartite motif (TRIM) protein family is recognized by the existence of three distinct domains: a coiled-coil region, a domain comprising B-box domains, and a RING finger domain.Notably, the Cterminal region of TRIM proteins exhibits significant variability among different family members [5].TRIM27 protein, a member of the TRIM protein family, is present in many human organs [6].In recent investigations, the oncogenic function of TRIM27 has been elucidated in diverse malignancies, including lung carcinoma, endometrial carcinoma, and breast carcinoma [7][8][9].The presented evidence demonstrates a consistent association between the TRIM27 expression and the natural progression of cancer.These findings suggest that this gene and its encoded products play a remarkable role in cancer development.
Genes METTL3, METTL14, and WTAP have been identified as key players in m 6 A methylation that can be reversed through m 6 A erasers, specifically the fat-mass and obesity-linked protein (FTO) together with AlkB homolog 5 (AlKBH5) [10][11][12].YTH domain family (YTHDF) together with heterogeneous nuclear ribonucleoproteins (hnRNPs) serve as m 6 A readers, enabling the recognition and subsequent regulation of downstream molecular mechanisms [13][14][15].YTHDF1 is a protein that comprises the YT521-B homology (YTH) domain.The recognition of m 6 A by this protein is facilitated by the conserved YTH domain, enabling it to modulate the functions of genes at the stage of post-transcription.This study has shown that the TRIM27 protein level was elevated in both CRC tissues and cells, depending on the m 6 A-YTHDF1 pathway.Overall findings indicate that TRIM27 is essential in maintaining CRC CSC-type characteristics.

CRISPR-Cas9 cell line formation
A lentivirus infection experiment was conducted to achieve stable gene knockout.The lentivirus used in this study was procured from Nanjing Jikai Gene Corporation.Approximately 100,000 cells were cultured within six-well plates overnight.The cell culture medium was supplemented with viral solutions based on the multiplicity of infection (MOI) to induce infection.Following an 8 h incubation, the medium was refreshed with a normal culture medium supplemented through 1 μg/ mL Puromycin (two-day exposure).Cultures were propagated, expended/validated via Western blotting (WB) analysis.

Formation of spheroids
Cultures undergoing logarithmic expansion were trypsinized/maintained within 10 % serum-harboring medium.Cells were resuspended within serum-free medium after centrifugation at 500 rpm/min.They were grown onto ultralow-attachment six-well plate (Corning™, USA), maintained in a culture medium consisting of 3 mL DMEM/F12, 20 mg/ ml human recombinant epidermal growth factor (Sigma Aldrich™, USA), 5 μg/mL insulin (Sigma-Aldrich™), and 2 % B27 (Invitrogen™, USA).Culture media were replenished at 72 h intervals.Cell imaging was performed via BioTek Cytation 5® cellular imaging multimode reader following a cell seeding of 7~10 days.Only spheroids with a diameter greater than 50 μm were quantified.

m 6 A RNA immunoprecipitation(RIP) assay
Total RNA was extracted from the respective CRC cells using TRIzol reagent (Invitrogen, Waltham, MA, USA).RIP was conducted with the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (#17-10499, Merck Millipore™, USA) according to the manufacturer's instructions.Enrichment of m 6 A was analyzed using RT-PCR analysis.

Clinical tissues
In this study, 12 patients have undergone surgical procedures at Longyan First Hospital, Affiliated with Fujian Medical University.Before the surgery, they were not exposed to chemotherapy or radiotherapy.6 patients had DDP-resistant (defined as tumour recurrence within six months after R0 excision during DDP-based therapy).6 patients had DDP-sensitive (defined as no tumour recurrence during DDPbased therapy).The harvested tissues were placed in liquid nitrogen and then cryopreserved at − 80 • C for subsequent examinations.Cases were segregated within separate distinct cohorts.The project received approval from the Institutional Review Board (IRB) of Longyan First Hospital, affiliated with Fujian Medical University.All procedures involving human participants in this study were conducted in accordance with the Declaration of Helsinki (as revised in 2013), and all patients or their respective families were provided with the study's rationale and obtained their consent prior to the participation.

m 6 A determination
Overall m 6 A RNA presence changes were quantified via EpiQuik m 6 A RNA Methylation Quantification Kit (Colorimetric) (#P-9005, Epigentek, Farmingdale, NY) in accordance with established guidelines provided by the manufacturer.The subsequent analysis required 200 ng of RNA from both tested CRC tissues and cell lines.

Western blotting
Cell lysates or tissue extracts were prepared following the previously established protocol [15].SDS-PAGE was performed to isolate all the proteins and transfer them onto 0.45 μm PVDF membranes (Millipore, IPFL85R).After blocking the PVDF membranes with TBS-T (5 % skim milk), followed by primary and secondary antibodies (1 h at RT), the proteins were visualized in accordance with the manufacturer's guidance via SuperSignal West Pico PLUS (Invitrogen,34,580).

Real-time(RT)-PCR
Total RNA was isolated from CRC tissues or cells using Trizol reagent (Invitrogen, Waltham, MA, USA), and then RNA was used to perform reverse transcription with a Transcriptor First Strand cDNA Synthesis kit (Roche, Basel, Switzerland).The acquired cDNAs were used as templates for real-time PCR analysis using SYBR Green PCR Master Mix (Qiagen, Valencia, CA, USA).The relative mRNA levels were calculated using the 2 − ΔΔCt method, with the levels normalized to GAPDH mRNA.

RNA lifetime assays
Cells were treated according to our experimental design.Actinomycin D (Sigma) was added at a concentration of 5 mg/mL.Then, at the indicated times, cells were lysed and the total RNA was extracted (Qiagen).RNA quantities were determined through q-PCR analysis.

Statistical analysis
One-way ANOVA analyzed the significance level of the disparity in all experiments, followed by post-hoc Dunnett's assessment.The logrank assessment determined the p-value for Immunohistochemical and microarray analyses.The cutoff for the significance level was p < 0.05.All experiments were executed in triplicates, and data were stated as the mean ± SD.

TRIM27 protein was increased in cisplatin (DDP)-resistant CRC
We firstly analyzed TRIM27 protein level in 6 cisplatin-sensitive and resistant CRC tissues.TRIM27 expression was substantially increased in DDP-resistant CRC tissues (N = 6) compared to DDP-sensitive CRC tissues (N = 6) (Fig. 1A).Meanwhile, we also found that TRIM27 protein level enhanced in the DDP-resistant HCT116 (HCT116/DDP) and SW620 (SW620/DDP) cells in comparison with control cells (Fig. 1B).The system of CRISPR/Cas9 was utilized to knockdown the expression of TRIM27 in the HCT116/DDP and SW620/DDP cell lines (Fig. 1C); nevertheless, TRIM27 knockdown failed to significantly affect the sensitivity of HCT116/DDP or SW620/DDP cells towards DDP treatment (Fig. 1D).These findings demonstrated that TRIM27 may not be necessary or implicated in DDP-resistant CRC, even though it was elevated in DDP-resistant CRC.

Role of TRIM27 in maintaining the CSCs-type characteristics in DDPresistant CRC
Spheroid formation assay exhibited that TRIM27 knockdown reduced the spheroid-forming capability of the DDP-resistant CRC cells (Fig. 2A and Fig. S1).RT-PCR analysis exhibited a substantial decrease in the expression of CRC stem-cell epitopes CD133/CD44 within HCT116/DDP or SW620/DDP cultures upon TRIM27 knockdown (Fig. 2B).Furthermore, flow cytometry analysis validated the downregulation of cellular superficial markers; CD133/CD44 within HCT116/DDP or SW620/DDP cultures following TRIM27 knock-down (Fig. 2C-D).These findings demonstrated that TRIM27 enhanced the CSCs-type characteristics in DDP-resistant CRC.

Upregulated TRIM27 translation in DDP-resistant CRC cells
Following, we investigated of the molecular mechanisms responsible for the TRIM27 upregulation in DDP-resistant CRC, and RT-PCR was executed to investigate the mRNA level of TRIM27 in DDP-sensitive and DDP-resistant CRC tissues and across different cancer cell lines.The findings revealed an insignificant disparity between DDP-resistant and sensitive CRC cells or tissues, suggesting that the TRIM27 expression is  regulated at the translation phase (Fig. 3A-B).Furthermore, the TRIM27 mRNA degradation exhibited comparable patterns in both DDP-resistant and control CRC cells (Fig. 3C).TRIM27 protein level was both upregulated in HCT116-control and SW620-control cells or HCT116-DDP and SW620-DDP cells under MG132 introduction, a potent proteasome regulator, or E64D/pepstatin A, lysosomal regulators, however, upregulated TRIM27 in HCT116-DDP and SW620-DDP compared to HCT116control and SW620-control cells were unchanged under the same condition treated(MG132 or E64D/pepstatin A) (Fig. 3D).These findings demonstrate that the TRIM27 mRNA and protein degradation remains unchanged in DDP-resistant CRC cells.Furthermore, TRIM27 mRNA was substantially elevated on monosomes and polysomes of DDP-resistant CRC cells relative to the control cells (Fig. 3E).In comparison to the DDP-sensitive CRC cells, GAPDH transcriptomic expression remained unchanged within DDP-resistant CRC cells(Fig.3F).Above findings suggested a potential involvement of translational activation in the upregulated of TRIM27 in DDP-resistant CRC cells.

YTHDF1 interaction with the TRIM27 mRNA and enhances its protein expression in DDP-resistant CRC cells
The m 6 A amending is identified by specific readers in translating target mRNAs (16).RIP analysis revealed that the TRIM27 mRNA level interaction with YTHDF1 was increased in HCT116/DDP and SW620/ DDP cell lines compared to the control cells, while the TRIM27 mRNA level interaction with additional m 6 A readers was consistent in HCT116/DDP and SW620/DDP cell lines compared to the control cells (Fig. 4A).Although DDP-resistant cells expressed YTHDF1 similarly to control cells (Fig. 4B), YTHDF1 knocked down reduced the protein TRIM27 level in HCT116/DDP and SW620/DDP cells (Fig. 4C).These results suggested that YTHDF1 may promote TRIM27 translation in DDPresistant CRC cells by interacting with its transcript.Subsequently, an investigation was also conducted to ascertain the potential alteration of m 6 A amending in the TRIM27 transcript.The application of MeRIP, utilizing a specific antibody targeting m 6 A amending, revealed noteworthy findings regarding the enrichment of TRIM27 3′UTR in HCT116/ DDP and SW620/DDP cells (Fig. 4D).These outcomes indicate a potential role of YTHDF1 in facilitating the TRIM27 mRNA translation through its ability to recognize m 6 A sites on the 3′UTR of the mRNA.To investigate the potential mechanism responsible for the m 6 A upregulation in the TRIM27 transcript, this study focused on examining the patterns of m 6 A writers or erasers.Surprisingly, this investigation revealed that the level of METTL3, METTL14, WTAP, ALKBH5, together with FTO did not show any notable variation when comparing control CRC cells to DDP-resistant cells (Fig. 4E).The quantification for global m 6 A RNA was conducted, revealing not notable variation between HCT116/DDP or SW620/DDP cells and their control cells (Fig. 4F).Meanwhile, TRIM27 mRNA elevated on monosomes and polysomes of DDP-resistant CRC cells relative to the control cells was impaired by YTHDF1 knocked down(Fig.4G).Additionally, the knockdown of YTHDF1 led to the corresponding suppression of CSCs-marker CD133, and CD44 expressions (Fig. 4H).These findings demonstrated that YTHDF1 interaction with the TRIM27 mRNA and enhances its protein expression in DDP-resistant CRC cells.

Discussion
The current findings presented evidence indicating an upregulation of TRIM27 in DDP-resistant CRC cells at the protein level, and TRIM27 knockdown remarkably reduced the expression of CSC resembling characteristics.Knock down TRIM27 inhibited the CSCs-type characteristics in DDP-resistant CRC Overall, indicating that TRIM27 is essential in maintaining CRC CSC-type characteristics.
The m 6 A amending is extensively recognized as a predominant occurring, widespread, and conserved amending in various RNA molecules.Its significance lies in its emerging role as a novel regulator of RNA processing, transcriptional regulation, alternative splicing, RNA stability, and translation control [16][17][18][19][20].The present investigation has revealed DDP-resistant tumors exhibit comparable levels for m 6 A amending compared to the control cells.There is a noteworthy upregulation in the m 6 A amending for TRIM27.However, dynamic and reversible m 6 A amending is primarily facilitated by specific enzymes known as writers (METTL3, METTL14, and WTAP) and erasers (FTO and ALKBH5) [21][22][23].No common amendings of m 6 A writers/erasers were identified within this study, which may explain the improvement of TRIM27 m 6 A amending in DDP-resistant CRC.Additional investigations are warranted to explore the precise mechanism(s) responsible for enhancing m 6 A amending for TRIM27 within DDP-resistant CRC.
The m 6 A amending remains identified through various readers, such as YTHDF1-3, YTHDC1-2, together with hnRNP family proteins [24][25][26].YTHDF1 can identify m 6 A post-transcriptional amending via a conserved aromatic cage upon YTH region [27][28][29].Despite the shared ability of YTH domain-containing proteins to interact with m 6 A transcriptomic locations, exhibiting distinct target mRNA recognition while performing different roles.Case in point, the translational efficiency of the mRNAs regulated by YTHDF1, YTHDF3, and YTHDC2, their stability was reduced by YTHDF2, YTHDF3, and YTHDC2, while YTHDC1 substantially involved in m 6 A mRNAs splicing and nuclear export [30].The present investigation has revealed that there is an enhancement in the recruitment of YTHDF1 to TRIM27 mRNA within DDP-resistant CRC cells.This heightened recruitment facilitated the translation of the TRIM27 transcript, While TRIM27 protein expression was considerably reduced by YTHDF1 knockdown.The existing studies indicate that m 6 A amending dysregulation is closely linked to the onset and advancement of diverse types of human cancers.Besides, m 6 A amending can act as either an oncogene or a tumor suppressor based on the specific characteristics of the cancer under consideration [31].Henceforth, the findings from this study strongly suggest that YTHDF1 exhibits promising effects as a viable therapeutic management of CRCs.

Conclusion
This study presented the involvement of TRIM27 as an oncogene that promotes the CSC-type properties of CRC cells.Further, YTHDF1, an upstream molecule for TRIM27, could identify delf 3′UTR, facilitating its translation within CRC.Hence, drawing upon the observed link between TRIM27 and the CSC-type properties in DDP-resistant CRC cells, TRIM27 holds promise as a possible therapeutic target for treating CRC.

Fig. 1 .
Fig. 1.Elevation of TRIM27 protein in cisplatin-resistant CRC A. Representatives immunoblot (left) and quantification (right) of TRIM27 protein level expression in DDP-resistant CRC tissues.B. Representatives immunoblot (left) and densitometric analysis (right) of protein expression of TRIM27 in HCT116 and SW620 and DDPresistant cells.C. Knocking down the TRIM27 expression via CRISPR-Cas9, and the efficacy was validated by Western blotting (left) and densitometric analysis (right).D. CCK8 analysis was used to evaluate cell viability in HCT116/DDP or SW620/DDP or TRIM27 (KD) cells treated with different concentrations of DDP for 48 h, *p < 0.05; N.S.not significant.

Fig. 2 .
Fig. 2. Role of TRIM27 in maintaining CSCs-type characteristics of DDP-resistant CRC cells.A.Spheroids formation and quantification from control or TRIM27-KD HCT116/DDP and SW620/DDP cells, B. Analysis of CD133 and CD44 expressions via RT-PCR.C-D.Flow cytometry analysis of CD133 (C) and CD44 expressions (D), and data were recorded via MFI (mean fluorescence intensity).*p< 0.05.

Fig. 3 .
Fig. 3. Upregulation of TRIM27 translation in DDP-resistant CRC cells.A-B.Analysis of TRIM27 mRNA level by real-time PCR within CRC tissue (A) or cellular cultures (B).C. Shortened RNA lifetime of TRIM27 mRNA in indicated CRC cell or CRC-DDP cells.D. Respective immunoblots (left) and quantification (right) of TRIM27 levels in HCT116 or SW620 cells treated with vehicle (SPECIFY VEHICLE USED), MG132 or E64D, and pepstatin A (one day).E. RT-PCR analysis for TRIM27 mRNA distribution, F. Real-time PCR analysis for GAPDH mRNA distribution.*p< 0.05; N.S.not significant.

Fig. 4 .
Fig. 4. YTHDF1 interaction with the TRIM27 mRNA and enhances its protein expression in DDP-resistant CRC cells.A. RIP evaluation via IgG or the YTHDF1-3, YTHDC1-2 antibodies, and RT-PCR analysis for TRIM27 mRNA enrichment.B. Analysis of YTHDF1 mRNA level by real-time PCR.C. Left panel:Knockdown YTHDF1 via shRNAs against YTHDF1 (shYTHDF1), and respective antibodies analyzed via Western blot, Right panel: Duantify densitrometric analysis.D. m 6 A RIP-PCR analysis by IgG or an m 6 A antibody.E. Real-time PCR analysis for METTL3, METTL14, WTAP, ALKBH5, and FTO mRNA expression.F.Quantification of total m 6 A RNA levels via ELISA.G. Real-time PCR analysis for TRIM27 and GAPDH mRNA distribution.H. Analysis of stem cell markers via RT-PCR.*p < 0.05; N.S.not significant.*p < 0.05; N.S. not significant.