The analysis of the circRNAs in the progress of acquired resistance to Cetuximab

We established a Cetuximab‐resistant cell lines by high‐dose pulse method and searched for differentially expressed circular RNAs by RNA sequencing. Hundreds of circRNAs were altered between sensitive and resistant cells. Next, we chose six notably differential circRNAs and conducted quantitative real‐time PCR by specific primers. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that differentially expressed circular RNAs are enriched in some tumor‐related pathways, such as tumor transcription regulation, metabolism, PI3K‐Akt, mTOR, and other signaling pathways. Our results explored differentially expressed circular RNAs associated with Cetuximab to find new targets for Cetuximab resistance therapy.


| INTRODUCTION
As one of the most common malignant tumors in the world, the global incidence rate of colorectal cancer (CRC) is estimated to be 10.0%, ranking the third and it is the second cause of cancer death (9.4%) in 2020. 1 In China, with the continuous changes in people's lifestyles and dietary habits, the incidence and mortality of CRC have been increasing year by year. The five-year survival rate of CRC patients is only 31%, which is far lower than that of developed countries such as the United States and Japan. 2 CRC has become an important problem that seriously endangers people health.
Noncoding RNAs (ncRNAs) have important roles in regulating the expression of genes that control fundamental biological functions. Compared with normal cells, altered levels of ncRNAs are frequently detected in cancer cells. Circular noncoding RNA (circRNA), covalently closed loops resulting from backsplicing of mRNA, is a new type of ncRNAs that is different from linear RNA. 3,4 It is widely present in various cells. Most of it comes from the exon region of genes, and a small part is formed by intron splicing. Many circRNAs exert important biological functions by binding and acting as sponges of miRNAs or protein inhibitors to regulate protein function. They have been implicated in diseases such as diabetes mellitus, neurological disorders, cardiovascular diseases, and cancers. 5,6 For instance, circRNA hsa_circ_0000523 was down-regulated in different CRC cell lines and suppressed apoptosis of CRC cells. In addition, it acted as a "sponge" of miR-31, indirectly regulating Wnt/βcatenin signaling pathway, which was involved in the progression of CRC. 7 circ 0006528 affects Raf1 through "sponge adsorption" miR-7-5p, thereby regulating breast cancer resistance to Adriamycin. 8 In this study, we cultured a Cetuximab-resistant cell lines by high-

| Validation of the Cetuximab-resistant cell line
The resistance index for H508/CR cells was determined by the CCK-8 assay. The IC50 values of H508/CR cells for Cetuximab was increased from 12.24 μg/ml for sensitive cells to 161.5 μg/ml. The resistance index was >10 ( Figure 1).

| Profiles of the differentially expressed circRNAs
The volcano plots and heatmap showed distinct expression patterns of the circRNAs varied within NCI-H508 and H508/CR cells by RNA-seq analysis (Figure 2A,B). Two hundred and eight mRNAs were significantly increased, while 211 were declined in Cetuximab resistant group.
KEGG pathways through bioinformatic tools revealed that significantly differentially expressed circRNAs are enriched in some tumor-related pathways, such as tumor transcription regulation, metabolism, PI3K-Akt, mTOR and other signaling pathways ( Figure 2C).

| DISCUSSION
As an effective epidermal growth factor receptor (EGFR) antibody, Cetuximab competitively binds to the extracellular domain of EGFR with the epidermal growth factor to treat CRC and can effectively reduce the overall mortality rate in CRC patients. 10,11 The acquired resistance to Cetuximab yet limits its application and causes tumor recurrence and metastasis. 12, 13 We do not fully understand the detailed mechanisms, expect for common genetic mutations.
High-throughput sequencing has been extensively used to identify differential RNAs including coding and noncoding RNAs that can be exploited for cancer treatment and drug development. 14,15 In view of the fact that circRNA related research has just started, the F I G U R E 2 (A) Volcano plot of the circRNA sequencing analysis revealed the des-regulated circRNAs. (B) Heatmap of the circRNA sequencing analysis. (C) KEGG pathway enrichment of markedly expressed circRNAs. In bubble charts, x-axis represents Rich factor, which is the ratio of the enriched differential circRNAs to the background; y-axis indicates KEGG pathways. Dot dimension represents enriched differential circRNAs quantity and its color indicates different p-value ranges relationship between Cetuximab resistance in CRC patients and cir-cRNAs has not been reported. Researchers performed RNA sequencing and qRT-PCR to explore differentially expressed circRNAs between CRC tissues and adjacent normal tissues. They highlighted circRHOBTB3 which is significantly decreased in both CRC tissues and cell lines. The higher the clinical stage and the risk of metastasis, the lower of its expression. 16 Furthermore, 50 CRC and 50 healthy control serum samples were involved in circRNAs investigations by RNA-seq analysis. 17 Previously, we study the relationship between Cetuximab resistance and long noncoding RNAs by establishing Cetuximab resistant cells. 9 At present, we chose six markedly expressed circRNAs and conducted qRT-PCR to confirm RNA-seq results. No reports before were concerned to some of these circRNAs. We further explore the new molecular target of Cetuximab resistance from the perspective of circRNAs, which is expected to reveal the new mechanism of drug resistance and provide new ideas for clinical treatment. It has positive theoretical significance and clinical application value.

AUTHOR CONTRIBUTIONS
Liping Yin wrote the manuscript and designed the study. Changwen Jing performed bioinformatic analysis. Yesong Guo contributed to the design of the study. All authors have approved the final manuscript.

This work was supported by scientific research project of Jiangsu
Commission of Health (M2020032).

CONFLICT OF INTEREST
There is no conflict of interests for all the authors.

ETHICS STATEMENT
The research did not involve human ethical issues.