M6A Promotes Colorectal Cancer Progression via Regulating the miR-27a-3p/BTG2 Pathway

Long noncoding (lnc) RNAs regulate cancer progression. However, the importance of lncRNAs and how they are regulated in colorectal cancer (CRC) are unclear. We aim to evaluate the function of lncRNA ADAMTS9-AS2 in CRC and its fundamental mechanism. Levels of ADAMTS9-AS2, miR-27a-3p, and B-cell translocation gene 2 (BTG2) were measured by qPCR. Cell viability was analyzed by CCK-8 and colony formation. Migration and invasion were tested by transwell assay. The interactions among ADAMTS9-AS2, miR-27a-3p, BTG2, and YTHDF2 were analyzed by luciferase test, immunoblotting, RNA pull-down, or RNA immunoprecipitation (RIP). An animal model was adopted to assess ADAMTS9-AS2's function. Overexpressing ADAMTS9-AS2 inhibited cell migration, invasion, colony formation capacity, and proliferation in vitro. The direct targeting of miR-27a-3p by ADAMTS9-AS2 abrogated the latter's effect in CRC cells. BTG2 was identified a target of miR-27a-3p, and silencing BTG2 weakened miR-27a-3p's effect. Knocking down ADAMTS9-AS2 abolished sh-YTHDF2's inhibitory effect on cell proliferation and invasion. Finally, overexpressing ADAMTS9-AS2 restrained xenograft growth. M6A reader YTHDF2-mediated degradation of ADAMTS9-AS2 promotes colon carcinogenesis via miR-27a-3p/BTG2 axis.


Introduction
Colorectal cancer (CRC) is a leading gastrointestinal system's malignancy, and a major reason of tumor-related deaths globally due to increased morbidity. Given the unclear symptoms of early CRC, almost 60% are diagnosed at the advanced stage [1]. A key reason for CRC death is tumor recurrence and metastasis, which is closely linked to migration [2]. Hence, it is critical to better understand CRC's progression and metastasis.
Long noncoding RNAs (lncRNAs) regulate gene expression through various mechanisms [3]. Several lncRNAs have been identifed in recent years that regulate tumor progression. A study reported that lncRNA HOTAIRM1 promoted thyroid cancer cells' growth and invasiveness [4]. Furthermore, data showed that the lncRNA FGD5-AS1 promoted chemoresistance of CRC cells [5]. ADAMTS9 is a tumor suppressor, and its antisense RNA 2 (ADAMTS9-AS2) transcript is a lncRNA that may impede tumor progression and metastasis [6]. Wang et al. reported that ADAMTS9-AS2 suppressed gastric tumor cell growth via regulating the expression of SPOP [7]. However, ADAMTS9-AS2's function in CRC is elusive.
Among malignant tumors, the incidence and mortality of lung cancer have always been among the top in the world. Lung cancer is histopathologically divided into nonsmall-cell lung cancer (NSCLC) and small cell lung cancer. About 85% of lung cancer patients have non-small-cell lung cancer. Advances in the diagnosis and treatment have helped to improve the survival of cancer patients; However, the 5year survival rate for NSCLC was 17.7. In addition, about 85% of patients with NSCLC are diagnosed at advanced stages. Terefore, further study of the pathogenesis of NSCLC, identifcation of new therapeutic targets, and prognostic biomarkers are the key to improve patient survival.
In this study, through bioinformatics analysis, the authors frst found that RMRP may play an important role in NSCLC. Further studies showed that m6A modifcation improved the stability of methylated RMRP transcripts by reducing the rate of RNA degradation. In addition, RMRP can promote cell proliferation, migration, and invasion. Mechanistically, RMRP promotes TGFBR1 transcription by recruiting YBX1 to the TGFBR1 promoter. Here, we investigated the function of ADAMTS9-AS2 in CRC progression, and its regulatory infuence of m6A modifcation. Data demonstrated that the novel ADAMTS9-AS2/miR-27a-3p/BTG2 ceRNA regulatory network might regulate CRC progression.

Patient Samples.
Seventy-eight paired tumor and adjacent colorectal tissues from CRC patients who underwent surgical resection between February, 2016, and February, 2019, at the 1st People's Hospital of Foshan were included. Patients underwent no radio-or chemotherapy. Informed consents were received. Tis research has the approval from Ethics Committee of Nanfang Hospital.

RT-qPCR.
RNAs were isolated using TRIzol (Invitrogen), and cDNA was synthesized with a kit (Takara). qPCR was done with SYBR-Green Mix (ABI). Te expression change was calculated by 2 −ΔΔCq [15]. Primers are shown as follows: Methylated RNA immunoprecipitation (MeRIP) is based on the principle of specifc antibody specifc binding to methylated modifed bases and on the basis of RNA immunoprecipitation enrichment of methylated modifed fragments, and then through high-throughput sequencing, the results were obtained by studying the RNA regions where methylation occurred on a transcriptome scale. m6A abundance was tested by EpiQuik. m6A RNA methylation quantitative kit (Biovision) was used for the m6A abundance test for the EpiQuik assay. In brief, 250 ng RNA was probed with m6A antibodies. Te immunoprecipitation was reverse-transcribed to cDNA, then m6A-lncRNA levels were measured by qRT-PCR.

Transwell Assay.
Transwell inserts (8 m, Costar, Corning) with (invasion assay) or without (migration assay) Matrigel (Matrigel Basement Membrane Matrix, Corning) coating were placed in 24-well plates. Cells were cultured in top chambers without serum at a concentration of 0.1 million cells/well (invasion) or 0.5 × 105 cells/well (migration), and bottom chambers were loaded with RPMI-1640 (10% FBS). Two days later, cells on the top surface were discarded, while cells traveled through membranes were fxed and counted.

RNA Pull-Down Assay.
Lysates of SW480 and HCT116 were incubated with biotin-labeled ADAMTS9-AS2 probe (RiboBio) and streptavidin-coupled magnetic beads. Proteins in the complex pulled down by ADAMTS9-AS2 were analyzed by immunoblotting.

Data
Analysis. SPSS 22.0 was adopted for analyzing data (mean ± SD). Comparisons between two or more groups were done by t-test or ANOVA. P < 0.05 was designated as signifcant.

ADAMTS9-AS2 Is Decreased in CRC.
Levels of ADAMTS9-AS2 in the CRC and normal colon tissues were analyzed using microarray data downloaded from TCGA database. Te heatmap revealed that ADAMTS9-AS2 was drastically downregulated in CRC tissues (Figure 1(a)). In the GEPIA datasets as well, ADAMTS9-AS2 in CRC was considerably decreased than normal colon tissues ( Figure 1(b)). Furthermore, patients with lower ADAMTS9-AS2 showed a shorter overall survival (OS) (Figure 1(c)). To verify the in silico data, we analyzed 78 pairs of CRC and adjacent tissues, CRC cells, and colon epithelial cells. ADAMTS9-AS2 was sharply suppressed in the CRC tissue ( Figure 1(d)) and cell (Figure 1(e)). Tus, ADAMTS9-AS2 is downregulated in CRC and portends a poor prognosis.

YTHDF2
Enhanced the Degradation of m6A-ADAMTS9-AS2 in CRC Cells. Te m6A demethylase FTO was overexpressed in the CRC cell lines. We therefore hypothesized that FTO may afect ADAMTS9-AS2 expression levels in CRC cells by altering its methylation status. As illustrated in Figure 4(a), overexpressing FTO in the SW480 and HCT116 cells signifcantly reduced m6A-ADAMTS9-AS2 levels. Furthermore, the hypomethylation of ADAMTS9-AS2 was related to a signifcant increase in its expression levels (Figure 4(b)), indicating that ADAMTS9-AS2 is regulated by m6A modifcation. In comparison to IgG immunoprecipitation, a RIP experiment demonstrated a higher concentration of ADAMTS9-AS2 in YTHDF2 immunoprecipitation (Figures 4(c) and 4(d)). RNA pull-down indicated that the complex pulled down by ADAMTS9-AS2 contained an     Journal of Oncology 7 abundance of YTHDF2 protein (Figures 4(e) and 4(f)). Te results revealed that YTHDF2 recognized ADAMTS9-AS2. YTHDF2 knockdown efectively reduced ADAMTS9-AS2 degradation (Figures 4(g) and 4(h)).

Discussion
Increasing evidence shows that aberrant lncRNAs are linked to the development of CRC. For example, ENO1-IT modulates KAT7 histone acetyltransferase and consequently altered CRC biological function [16]. Furthermore, LINC00265 is upregulated in CRC, and its knockdown in mice signifcantly reduced colorectal carcinogenesis [17]. We found that ADAMTS9-AS2 was decreased in the CRC samples in TGCA database, and associated with the worse survival rate. ADAMTS9-AS2 (Ensembl, ENSG00000241684) has been linked to several tumor-associated genes in multiple cancers. For example, ADAMTS9-AS2 was increased in TMZ-resistant glioblastoma cells to enhance chemoresistance by upregulating the FUS/MDM2 axis [18]. We also demonstrated that ADAMTS9-AS2 decreased in CRC. Decreased ADAMTS9-AS2 was linked to poor diferentiation, lymph node metastases, and advanced TNM staging. Overexpressing ADAMTS9-AS2 in CRC cells inhibited their malignant potential in vitro. Tus, ADAMTS9-AS2 is a potential marker for the CRC prognosis and may function as a tumor suppressor. Te hypothesis that ceRNA (competitive endogenous RNA), proposed by Pier Paolo Pandolf's group at Harvard Medical School in 2011, is a mode of regulating gene expression. Transcripts that share miRNA-binding sites compete to bind the same miRNA, thereby regulating each other's expression levels. Based on ceRNA hypothesis, lncRNAs regulate mRNAs posttranscriptionally by competitively binding to miRNAs containing response regions [19,20]. For instance, lncRNA UCA1 sponges miR-143 and upregulates MYO6, thereby promoting CRC [21]. Likewise, UICLM promotes CRC metastasis by upregulating ZEB2 via its sponging action on miRNA-215 [22]. A previous study showed that miR-27a-3p was upregulated in CRC, and silencing it decreased cell growth [12]. BTG2 was proved as a miR-27a-3p's target by the luciferase assay and RIP assay. BTG2 regulates cell division, DNA repair, transcriptional control, and mRNA stability [23].
BTG2 was decreased in diferent cancers. For instance, BTG2 decrease promoted breast cancer's metastasis [24]. Likewise, miR-6875-3p afects cancer cells' invasiveness via BTG2 [25]. We revealed that BTG2 was decreased in CRC, and its silencing abrogated miR-27a-3p's pro-oncogenic efects. Given the abundance of m6A modifcation in eukaryotic mRNAs, it has received considerable attention as a regulatory factor in cancer and other pathological and developmental states.
Studies demonstrated that m6A binding protein YTHDF2 destabilized EGFR via binding to m6A site, and inhibited hepatocellular carcinoma cells [26]. YTHDF2 also regulates the stability of lncRNAs and mRNA during cancer development. Another study revealed that YTHDF2 inhibited lncRNA GAS5 in cervical cancer cells by promoting its degradation [27]. Consistent with this, hypermethylation of ADAMTS9-AS2 increased its degradation through the recruitment of YTHDF2. Tese fndings show that aberrant m6A modifcation of ADAMTS9-AS2 is reliant on YTHDF2. Knocking down YTHDF2 inhibited CRC cell proliferation and invasion by restoring ADAMTS9-AS2 expression, implying that YTHDF2 may have an oncogenic role in CRC.

Data Availability
Te experimental data used to support the fndings of this study are available from the corresponding authors upon request.

Disclosure
Wenjun Liu and Zilang Zhang are the co-frst authors.

Conflicts of Interest
Te authors declare that they have no conficts of interest regarding this work.

Authors' Contributions
WJL and LXT supervised the project. WJL, KQ, BJH, and JZD carried out experiments. ZLZ and CYY collected data and performed analysis. WJL and LXT wrote and edited the manuscript. Wenjun Liu and Zilang Zhang contributed equally to this paper.