N6-Methyladenosine Modication of PTTG3P Contributes to Colorectal Cancer Progression via IGF2BP2

Background N6-methyladenosine (m 6 A) and long noncoding RNAs (lncRNAs) emerged as crucial players in colorectal cancer (CRC) progression, but the m 6 A modied lncRNA PTTG3P in CRC are still need to be systematically dened. Methods qRT-PCR was adopted to measure the PTTG3P expression. Survival analysis was used to explore the correlation between the expression of PTTG3P and CRC patients prognosis. Receiver operating curve (ROC) was tested to evaluate the PTTG3P predictive ability. Functional studies were examined by CCK-8, glucose uptake, lactate assay, ATP assay, ECAR assay and xenograft mice model. Mechanistic studies were explored by GSEA, methylated RNA immunoprecipitation sequencing (MeRIP-Seq) and RNA immunoprecipitation (RIP).


Background
Colorectal cancer (CRC) remains one of the major triggers of deaths from malignant tumors. Globally more than 1 million people suffer CRC every year [1] . As of 2012, CRC is the fourth cause of cancer death after lung, stomach, and liver cancer [2] . Despite improvements in diagnosis and combined treatment, patients with CRC have an even worse prognosis, especially in advance patients. Therefore, it is quite urgent to clarify the mechanism, even potential approaches for the therapeutic intervention of CRC.
Accumulating evidence has shown that pseudogene, a type of long noncoding RNA, exhibites pivotal functions. It is estimated that human genome has more than 18,000 pseudogenes. And pseudogene has emerged as key regulators of important biological processed involved in the development of human cancers. For instance, increased CYP4Z1 expression promotes tumor angiogenesis and growth in breast cancer partly via PI3K/Akt and ERK1/2 activation [3] . Nanog regulates primitive hematopoiesis by directly repressing critical erythroid lineage speci ers [4] . PTENP1 can exert a growth suppressive role by regulating cellular levels of PTEN [5] . LncRNA PTTG3P (pituitary tumor-transforming 3, pseudogene, NR_002734), located at chromosome 8q13.1, was rst reported in the study of human pituitary tumor transforming gene (hPTTG) family in 2000 [6] . However, its biological function of Warburg effect has yet to be illustrated in CRC. N6-methyladenosine (m 6 A) depicts the methylation of the nitrogen at position 6 in the adenosine base within mRNA. N6-Methyladenosine (m 6 A) was originally characterised in the 1970s [7] . Nowadays, the linkages between m 6 A and malignant tumors have been indicated, including breast cancer, prostate cancer, pancreatic cancer, kidney cancer, eukaemia, stomach cancer, sarcoma, and leukaemia [8][9][10][11][12] .
Our data discovered that highly expressed PTTG3P, induced by METTL3, predicts poor prognosis in patients with CRC. Further study revealed that PTTG3P could facilitate proliferation and glycolysis by regulating METTL3/PTTG3P/YAP1 axis. This research might provide a prospective therapeutic target for CRC treatment.

Statistical analysis
All the data were showed as the mean ± standard deviation, at least three independent experiments. Data were compared using the chi-square (χ2 test), Student's t-test or one-way analysis of variance (ANOVA). ROC curve was applied to evaluate the predictive ability of CRC diagnosis. Univariate and multivariate analysis were performed to ascertain independent factors for the diagnosis of CRC. Kaplan-Meier method was applied to measure the overall survival (OS). SPSS 22.0 (SPSS Inc., Chicago, IL, USA) was used to conduct statistical analyses, and differences were ensured when P-value was < 0.05.

PTTG3P is highly expressed in CRC
To evaluate potential lncRNAs involved in mediating CRC progression, we examined the lncRNA expression pro le (GSE 84983) ( Figure S1a). Comparison between CRC tumor tissues and adjacent normal tissues, we focused on the upregulated lncRNAs (fold change > 5, P < 0.01), for these lncRNAs might be oncogenes and therapeutic targets. LncRNA PTTG3P was one of the most upregulated and chosen for consideration ( Figure S1b). Then, we found that PTTG3P had rarely ability to code proteins, using the open-reading frames (ORFs) Finder and conserved domain database. Moreover, ve other different online metrics got the same conclusion (Table S3). Additionally, we identi ed no valid Kozak consensus sequence in PTTG3P [13] , indicating that PTTG3P is a long noncoding RNA with no proteincoding potential.
To verify the elevation of PTTG3P in CRC, we investigated the detailed annotative process of preclinical human cancer models via the Cancer Cell Line Encyclopedia (CCLE) (www.broadinstitute.org/ccle), indicating that PTTG3P is remarkably overexpressed in cell lines of CRC (Fig. 1a, 1b). Then, the cell lines of HT29, HCT-8, SW480, HCT-116 and FHC (human normal colorectal mucosal cell ) were conducted for PTTG3P expression. As showed in Fig. 1c, the PTTG3P expression was exceedingly increased in HT29, HCT-8, SW480 and HCT-116 cells, compared with FHC cells.
Further, we explored PTTG3P expression in a cohort of 60 paired and non-tumor tissues of CRC, the clinicopathologic characteristics are demonstrated in Table 1. Signi cantly, the PTTG3P level was overexpressed in CRC tissues compared to their counterparts (Fig. 1d, 1e), which was in accordance with the results of TCGA database (Fig. 1f, 1g). Besides, high PTTG3P expression was observed in many kinds of tumors compared with normal counterparts (Fig. 1h). Also, our specimens con rmed PTTG3P overexpression in stomach adenocarcinoma (STAD), and esophageal squamous cell carcinoma (ESCA) (Fig. 1i,1j). All together, these data revealed that PTTG3P was elevated in CRC and might be an oncogene. To identify the connection between the level of PTTG3P and clinicopathologic features, we divided the cases into PTTG3P low-expression and high-expression group on the basis of median expression. Upregulated PTTG3P was positive linked with Tumor size (P = 0.02) and Differentiation (P = 0.01), but not with age (P = 0.86 ), gender (P = 0.74), tumor invasion depth (P = 0.28 ), lymph node metastasis (p = 0.09) or vessel invasion (P = 0.06) ( Table 2). Moreover, the PTTG3P expression was higher in stage III-IV (advanced stage) than stage I-II (early stage) in tissues (Fig. 2a). Additionally, Kaplan-Meier survival curves illustrated that patents with highly expressed PTTG3P had poorer survival time (Fig. 2b). Further, we determined the prognostic ability of PTTG3P in CRC. As shown in Table 3, univariate analyses suggested highly expressed PTTG3P was associated with a dramatic risk of death (P < 0.01). Multivariate analysis demonstrated that PTTG3P expression could be an independent prognostic factor (P < 0.01). Subsequently, ROC curve was carried out to evaluate the diagnostic value of PTTG3P in CRC tissues compared with normal counterparts, the area under the ROC curve (AUC) was 0.776 (95% CI 0.733-0.819) (Fig. 2c). Thus, these data suggested that high expression of PTTG3P predicted a worse prognosis and may serve as a clinical biomarker for CRC patients.

PTTG3P promotes glycolysis and proliferation in CRC
To investigate the biological function of PTTG3P, we respectively transfected the PTTG3P overexpressed plasmids and silenced shRNA targeting PTTG3P into HT-29 and HCT-116 cells,respectilvely (Fig. 2d). By determining PTTG3P expression via gene set enrichment analysis (GSEA) the Cancer Genome Atlas (TCGA) pro les, we found that PTTG3P levels were positively correlated with the glycolysis by affecting genes in glycolysis regulation (Fig. 2e). To verify results of this analysis, PTTG3P knockdown restrained the mRNA level of GLUT-1, ALDOA, PKM2 and LDHA. Intriguingly, the effect of sh-PTTG3P on glycolytic gene transcription could be rescued by PTTG3P re-expression (Fig. 2f). Next, we performed the glucose uptake analysis, ATP analysis, lactate production analysis, and discovered that sh-PTTG3P repressed these. In contrast, PTTG3P overexpression boosted the glucose uptake (Fig. 3a), lactate production ( Fig. 3b), and ATP accumulation (Fig. 3c). Additionally, we calculated the level of ECAR, sh-PTTG3P notably repressed glycolytic capacity and vice verse (Fig. 3d). Also, we found that silenced PTTG3P suppressed the proliferation and facilitated apoptosis of HCT-116 cells, whereas upregulated PTTG3P increased the proliferation and inhibited apoptosis of HT-29 cells according to the CCK-8 assay and ow cytometry analysis (Fig. 3e,3f). In vivo, highly expressed PTTG3P e ciently increased the tumor growth ( Fig. 3g,3h). We then explored whether glycolysis plays a vital role in PTTG3P modulation of cell proliferation and tumor growth. Notably, the glycotic inhibitors 2-DG and 3-BP or depletion of LDHA, which could catalyze the nal step of glycolysis, could partly abrogate cancer cell proliferation and tumor growth (Fig. 3i,3j,3k).
Clinically, oxaliplatin is used for treatment of colorectal cancer. Previously, it is reported that suppression of glycolysis is an effective strategy to block cell proliferation and conquer drug resistance. Hence, we speculated that PTTG3P ablation in combination with oxaliplatin could strikingly repress tumor growth. As shown in Fig. 3l,3m, PTTG3P depletion could be conducted simultaneously with oxaliplatin. As a taken, PTTG3P knockdown plus oxaliplatin is a promising therapy for CRC.

PTTG3P regulates Hippo signaling pathway in CRC
In order to elucidate which pathway involved in PTTG3P-meddated CRC progression, GSEA in the published TCGA CRC database was explored. And we suggested that PTTG3P expression was associated with the YAP1-activated gene signatures, indicating that Hippo signaling pathway may participated in the biological function of PTTG3P (Fig. 4a). To verify the speculation, the hub genes in Hippo pathway, including LATS1/2, MST1/2 and YAP1, and Hippo pathway target genes, such as CDX2, FOXM1, CTGF and CYR61, were tested in sh-PTTG3P HCT-116 cells. Subsequently, PTTG3P knockdown impaired the mRNA level of YAP1, FOXM1 and CTGF (Fig. 4b).
It is commonly acknowledged that YAP1, a crucial factor in Hippo pathway, involves in cell proliferation and suppressed apoptotic genes. In our study, the level of PTTG3P and YAP1 displayed positive linkage in CRC tissues (Fig. 4c). And the association between YAP1 expression and clinicopathologic characteristics from TCGA indicated in table S4.

m 6 A modi cation is involved in the overexpression of PTTG3P in CRC cells
We next explored the upstream factors for PTTG3P elevation in CRC. Our study found no in uence on PTTG3P expression using DNA methyltransferase inhibition (Fig. 5a). Accumulating evidence has shown that ectopic expression of lncRNAs could be regulated by transcriptional factors, and histone acetylation plays a critical role in this procession. Then, we explored whether histone acetylation exerted a role in PTTG3P expression using SAHA and NaB, the broad-spectrum HDAC inhibitors, and we discovered that these HDAC inhibitors failed to alter PTTG3P level in HT29 cells (Fig. 5b). Further, overexpressed HDAC6 and HDAC8 had no effect on increasing PTTG3P expression (Fig. 5c). Subsequently, MeRIP-qPCR discovered that the m 6 A modi cation expression was dramatically increasing in the CRC cells compared with normal cells (Fig. 5d). Then, we con rmed that METTL3, a writer of RNA modi cation, signi cantly elevated the PTTG3P expression in both HT-29 and HCT-116 cells (Fig. 5e). Besides, overexpressed ALKBH5, an eraser of RNA modi cation, greatly suppressed the PTTG3P expression (Fig. 5f). Moreover, we conducted RNA stability analysis by treating cells with Act-D, binding DNA at the initiation complex and preventing RNA chain elongation, our ndings uncovered that highly expressed METTL3 strengthened the stability of PTTG3P (Fig. 5g). Therefore, m 6 A modi cation acts as an crucial factor in PTTG3P expression. As recently reported, Insulin-like growth factor-2 mRNA-binding proteins 1, 2, and 3 (IGF2BP1-3) are described as a type of m 6 A readers. We then evaluated the potential binding of PTTG3P and IGF2BP1-3, RIP-PCR was performed using an antibody against IGF2BP1-3. The results found that METTL3 overexpression increases binding between PTTG3P and IGF2BP2 in both HT-29 and HCT-116 cells (Fig. 5h). Interestingly, IGF2BP2 knockdown could partly abrogate the ability of Mettl3 upregulating PTTG3P (Fig. 5i). Finally, the association between METTL3,IGF2BP2 expression and clinicopathologic characteristics from TCGA indicated in table S5,S6.
Additionally, METTL3/PTTG3P high and PTTG3P/YAP1 high group certi ed an unsatisfactory prognosis than low group (Fig. 6g,6h). We further found that higher levels of METTL3, ALKBH5, and IGF2BP2 predicted poor prognosis and diagnostic value in CRC ( Figure S2).

Discussion
Pseudogene may be transcribed into RNA at low levels, due to promoter elements inherited from the ancestral gene or arising by new mutations. Although most of transcripts have rarely functional signi cance than chance transcripts from other parts of the genome, some have given rise to regulatory RNAs and new proteins. For instance, long noncoding RNA HK2P1, a pseudogene of HK2, promoted the lactate production and glucose uptake in endometrial stromal cells [14] . Pseudogene PTENP1 repressed the oncogenic PI3K/AKT pathway and inhibited the HCC progression [15] . To date, the role of pseudogene PTTG3P in glycolysis of CRC has not been reported yet. Our ndings documented that PTTG3P facilitated CRC progression via a METTL3/PTTG3P/YAP1 axis.
Our study veri ed that PTTG3P is highly expressed and has a potential diagnostic value, with an AUC of 0.776 (95% CI 0.733-0.819) in CRC. Clinically, high PTTG3P expression considerably associate with tumor size and TNM stage as well as shorter survival time. These results con rmed that PTTG3P overexpression serves as a valuable prognostic biomarker and aids innovatively e cient therapies for CRC patients. Additionally, our ndings stands in line with other research, Liu,et al. [16] reported that PTTG3P was remarkably upregulated in CRC tumor samples than that in normal samples. Zhou, et al. [17] revealed that PTTG3P is a valuable resource for identi cation in HCC progression and is useful for biomarker development. Weng, et al. [18] certi ed that PTTG3P facilitates cell proliferation, migration and invasion and might serve as a new promising strategy for gastric cancer. Recently, PTTG3P expression has a relationship with breast cancer [19] and pancreatic cancer [20] . Thus, the oncogenic role of PTTG3P in malignant tumors is strongly suggested.
Malignant tumors could undergo glycolysis at a higher speed than that of non-tumor tissue controls [21][22][23] . This phenomenon is known as Warburg effect [24] . The Warburg hypothesis demonstrates that malignant tumor is fundamentally caused by mitochondrial metabolism disorder. Doherty JR, et al. [23] found that tumor lactate levels correlate with increased metastasis, tumor recurrence, and poor outcome. And targeting lactate metabolism is a prospective method for cancer therapeutics. Furthermore, cancer cells with high level of glycolysis and acid resistance have a energetic growth advantage, which facilitates unrestrained proliferation and invasion. In our study, we explored gain and loss-of-function approaches in HT29 and HCT-116 cells and found PTTG3P ablation resulted in the inhibition of CRC cell glycolysis by regulating numerous genes linked with metabolic pathways, whereas the opposite outcome was observed after enforced expression of PTTG3P. Nowadays, ketogenic diet was used to constrain glycolysis to starve cancer cells, adjusting mitochondrial metabolism [25] . Here, we also proposed that the biological mechanism of PTTG3P on boosting cell proliferation might resist to apoptosis.
Hippo signaling pathway has become increasingly important in human cancer [26] , the key regulator YAP1 has been certi ed to be upregulated in breast cancer, colorectal cancer, and liver cancer [27] , and YAP1 could promote cell growth [28][29][30] and inhibit apoptosis [31] . Clinically, YAP1 could be a target for the development of cancer drugs [32] . Yi, et al. [33] suggested that inhibiting TEAD-YAP1 interactions or block the binding function of WW domains is a pharmacologically viable strategy against the YAP1 oncoprotein. In our presented study, we discovered that PTTG3P activates Hippo signaling pathway by promoting YPA1, FOXM1 and CTGF, not MST1/2, and rescue assay consolidates this by using Hippo pathway inhibitor, XMU-MP-1 (inhibiting MST1/2).
As recently reported, many studies have certi ed the important roles of METTL3 in the RNA degradation and stability. METTL3 (m 6 A methyltransferase ) and ALKBH5 (m 6 A demethylases) coordinately mediate the m 6 A modi cation of mRNA level of CDCP1, and m 6 A reader YTHDF1 could identify the m 6 A residues on CPCP1 mRNA 3′-UTR, which is installed by METTL3 to stimulate the CDCP1 translation [34] . METTL3 de ciency causes cancer cell apoptosis and repress cancer cell invasion [35] , while the activation of ALKBH5 by hypoxia was found to induce cancer stem cell enrichment [36] . Our ndings demonstrated that m 6 A methylation de nitely take part in PTTG3P upregulation, by enhancing its transcript stability. Moreover, We revealed that overexpressed METTL3 could enhance the binding between IGF2BP2 and PTTG3P, because of m 6 A-induced RNA structure alteration. Interestingly, IGF2BP2 depletion could partly abrogate the ability of Mettl3 upregulating PTTG3P.

Conclusion
In conclusion, our study rst discovers that METTL3/PTTG3P/YAP1 axis promotes Warburg effect in CRC, and m 6 A readers IGF2BP2 participates this progress. Hence, PTTG3P abalation might be used as a signi cant target for CRC prevention and therapy, shedding some light on the poorly understanding of The datasets used and analyzed in the current study are available from the corresponding author on reasonable request.

Competing Interests
The authors declare that there are no competing interests associated with the manuscript.

Funding
Supported by Natural Science Foundation of Liaoning Province of China.

Author Contributions
The work presented here was carried out in collaboration between all authors. Yue wang and guohua zhao contributed to the conception of the study; yang zheng contributed signi cantly to analysis and manuscript preparation. Guilin yu performed the data analyses and wrote the manuscript; longfei xie helped perform the analysis with constructive discussions.

Statement of Ethics
Patients have given their written informed consent in our study. And ethics committee of the rst hospital of dandong and liaoning cancer hospital approved the study protocol.

Consent to Publication
All the authors agreed to publish the manuscript.