1Department of Gastrointestinal Hernia, Ganzhou People’s Hospital, Ganzhou, Jiangxi, 341000, People’s Republic of China; 2Department of Hepatobiliary and Pancreatic Surgery, Ganzhou People’s Hospital, Ganzhou, Jiangxi, 341000, People’s Republic of China

Correspondence: Rongqiang Ye, Department of Hepatobiliary and Pancreatic Surgery, Ganzhou People’s Hospital, No. 16 Meiguan Avenue, Zhanggong District, Ganzhou, Jiangxi, 341000, People’s Republic of China, Tel +86-07975889858, Email [email protected]

Background: The diversity of available chemotherapeutic modalities for colorectal cancer (CRC) entails the implementation of personalized therapeutic regimens to optimize patient outcomes. Currently, the clinical use of biological markers for treatment selection is inadequate to achieve individualization. Circulatory RNAs (circRNAs), which function as plasma biomarkers, play a critical role in regulating biological processes in different types of cancer.
Methods: The samples (serum) were obtained from 80 CRC patients and 80 healthy individuals (controls) to assess the level of hsa_circ_0023919 via qRT-PCR analysis.
Results: In findings, hsa_circ_0023919 has a positive association with the disease stage and is greatly elevated in chemoresistant CRC patients. In addition, the area under the curve for hsa_circ_0023919 was modest, and an increase in hsa_circ_0023919 expressions was linked with a decreased overall survival (OS) and progression-free survival (PFS). Serum hsa_circ_0023919 levels serve as a diagnostic indicator for chemoresistance in CRC.
Conclusion: The findings suggested that hsa_circ_0023919 contributes to promoting chemoresistance in CRC patients. Consequently, it can be considered a potent therapeutic target for CRC treatment.

Introduction

Globally, CRC is listed as the fourth most prevalent cause of cancer-related fatalities.1 Notably, a new subgroup of unknown cancers of undefined origin (CUP) with good prognosis has recently emerged, which is clinically regarded as CRC, which will help to increase the incidence rate of CRC at present.2 Due to the development of multidisciplinary therapeutics, the median overall survival (OS) for CRC has remarkably increased over the last two decades, from 6–12 months to over 30 months.3,4 Older CRC patients are more likely to develop severe postoperative complications, but age has no effect on the relative survival rate of T4 stage CRC patients.5 Various cytotoxic agents, including anti-angiogenesis inhibitors, irinotecan, anti-epidermal growth factor (EGFR), and fluoropyrimidine (FP), are employed in first-line chemotherapy.6

Currently, tumor locations and RAS mutations serve as prognostic indicators for the efficacy of anti-EGFR antibodies, and BRAF mutations are applied as a clinical indicator of poor prognosis, as suggested by the European Society for Medical Oncology (ESMO) recommendation.7 Consequently, selecting an appropriate treatment for each patient is critical; moreover, the current clinical implementation of indicators for treatment selection needs to be improved. Thus, the discovery of predictive biomarkers is expected to facilitate treatment decisions that contribute to the improvement of CRC patient management.8 The KRAS, NRAS, and BRAF genes or aberrant miRNA may serve as biomarkers for CRC, although an optimal detection and validation strategy is still needed.9 The identification of indicators that can predict treatment responses and resistance to the available therapies may facilitate the decision of the highly effective first-line chemotherapeutics for CRC. Biomarkers capable of quantifying the extent of chemoresistance are likewise regarded as valuable tools in the development of suitable treatment approaches, such as adjustments to the treatment. The detection of these indicators would aid in the understanding of the role of the acquisition of resistance and the development of interventions. The elucidation of the process underlying acquired resistance might act as a basis for the development of drugs for overcoming the resistance.

Circular RNAs (circRNAs), which belong to the family of non-coding RNAs, are abundant in mammalian cells and have been identified in exosomes, body fluids, and tissues.10,11 Recently, an increasing body of research has unveiled the discovery that mammalian genomes contain tens of thousands of circRNAs, the dysregulation of which has been the subject of extensive investigation in relation to the development and progression of malignancies.12,13 However, additional research is required to determine whether newly identified circRNAs exhibit biological functions in malignancies.

The predominant function of circRNAs in many cancers has been confirmed as the “miRNA sponge” by data from previous studies. Employing sponging miR-140-3p,14 Hsa_circRNA_0088036 functions as a ceRNA and enhances the progression of bladder cancer. In sponging miR-145 and modulating NRAS,15 CircRNA_0044556 reduces the susceptibility of triple-negative breast cancer cells to adriamycin. Some circRNAs have been linked to CRC chemoresistance based on a number of previous studies.16,17

The plasma expression patterns of specifically altered circRNAs may function as prognostic or diagnostic indicators.18,19 The gene symbol of hsa_circ_0023919 is PICALM, and PICALM is reported to act as a pro-oncogene in CRC.20 It has been noted that serum hsa_circ_0023919 has been implicated in amyotrophic lateral sclerosis as a possible blood biomarker.21 However, there is currently no information regarding the possible role of hsa_circ_0023919 in serum as a mediator of chemoresistance in CRC patients. One of the most frequently used first-line chemotherapeutics for CRC patients is combination therapy involving bevacizumab (BEV), FP, and OX. In this study, hsa_circ_0023919 was selected as the prognostic indicator for FP+OX+BEV therapy. Quantitative qRT-PCR was employed to examine the correlation between chemoresistance and the clinical use of hsa_circ_0023919 in CRC patients who were treated with FP+OX+BEV.

Materials and MethodsSample Collection

In the present study, samples were collected from 80 CRC patients who were treated with the FP+OX+BEV treatment as the initial course of chemotherapy. A CT examination was performed on all patients every three months following chemotherapy to assess chemo-efficacy as per the Response Evaluation Criteria in Solid Tumors.22 In this study, responders were defined as those who obtained a full response, partial response (PR), or reduced stable disease (SD). Non-responders were those who experienced progression disease (PD) or prolonged SD. The Ganzhou People’s Hospital gave approval for such an investigation, conducted in line to the Declaration of Helsinki. Investigation participants submitted written informed-consent for specimen gathering.

Rt-Pcr

RNA was isolated from the plasma samples in line with the manufacturer’s instructions via the QIAGEN miRNeasy Serum/Plasma Advanced Kit (QIAGEN, Dusseldorf, Germany). The GENEJET RNA purification kit (Fermentas, Lithuania, USA) was utilized to extract RNA from the tissues of the patients as per the recommendation by the manufacturer. Using the Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, USA), cDNA was reverse-transcribed from RNA in a 20 µL reaction mixture. qRT-PCR was conducted on an Applied Biosystems ABI Viia7 (Thermo Fisher Scientific, Waltham, USA) utilizing the AceQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). The 2−∆∆Ct method was employed to determine the levels of circRNAs, with GAPDH serving as the control.

Statistical Analysis

Statistical analysis was performed via GraphPad Prism 8.0 (GraphPad, Inc., CA, USA). The data were compared, as appropriate, via Student’s t-tests and Mann-Whitney tests. A correlation between the plasma circRNA levels and clinical features was examined by applying the chi-square test. Binary logistic regression analyses were performed in the development of the hsa_circ_0023919 diagnostic model. ROC curve analyses were utilized to identify the most effective cutoff values for plasma circRNA expression, thereby increasing the diagnostic value after ROC curve formation via GraphPad Prism 8.0. The statistical level of significance was represented as a P-value ≤ 0.05.

ResultsPatients Characteristics

The detailed information of the patients who enrolled in this study is listed in Table 1. In this study, approximately 100 participants were recruited, consisting of 38 males and 42 females, and 55 patients were over 60 years old and 25 patients were under 60 years old. There were no significant differences in terms of age, sex, or other demographic data between the CRC patient and volunteer.

Table 1 Analysis of the Connection Between Characteristics of Responder and Non-Responder

CRC Patients Exhibit Elevated Levels of Serum Hsa_circ_0023919

To evaluate the efficacy of hsa_circ_0023919 as a marker of chemoresistance, its concentration in the serum was measured. There was a remarkable increase in serum hsa_circ_0023919 expressions among CRC patients relative to the control group (Figure 1A). Moreover, its expression was higher in patients with chemoresistance (n = 46) as opposed to those with chemosensitivity (n = 34) (Figure 1B).

Figure 1 CRC patients revealed high levels of serum hsa_circ_0023919. (A) Serum hsa_circ_0023919 levels were elevated in CRC patients. (B) Chemoresistant patients displayed elevated levels of hsa_circ_0023919 expressions relative to chemosensitive patients in post-treatment conditions. *p ≤ 0.05.

Association Between Hsa_circ_0023919 Levels and Clinical Manifestations of CRC Patients

The CRC patients were arranged into high and low groups of hsa_circ_0023919 levels in serum, based on the normal expression. In addition, clinical symptoms among both groups were examined. Chi-squared tests revealed a substantial relationship between hsa_circ_0023919 expressions and histological grade, tumor size, and TNM stages (Table 2). Conversely, age, gender, lymph node metastasis, lymphatic invasion, venous invasion, liver metastasis and peritoneal dissemination exhibited a no significant link with hsa_circ_0023919 levels. The Kaplan–Meier assessment indicated that patients with decreased levels of hsa_circ_0023919 have a prolonged OS as opposed to those with increased expression levels (Figure 2).

Table 2 Comparative Characteristics of Patients with High and Low Expression Levels

Figure 2 Expressions of hsa_circ_0023919 are linked with the clinical manifestations of CRC patients. Patients who showed elevated hsa_circ_0023919 expression displayed substantially greater OS relative to those with decreased levels.

Chemoresistant CRC patients with Hsa_circ_0023919 Exhibit a Poor Prognosis

Chemoresistant CRC patients have substantially reduced PFS (Figure 3A) and OS (Figure 3B) than chemosensitive patients, as shown by the Kaplan–Meier and Log rank tests. The significance of this circRNA as a distinct predictor of survival in chemoresistant CRC patients was highlighted by Cox proportional hazards regression analyses that revealed a correlation between hsa_circ_0023919 levels and histological grade, tumor size, TNM stages, and chemoresistance based on patient PFS (Table 3) and OS (Table 4).

Table 3 Two Types of Analyses (Univariate and Multivariate) of the Progression-Free Survival of CRC Patients

Table 4 Two Types of Analyses (Univariate and Multivariate) of the Overall Survival of CRC Patients

Figure 3 Chemoresistant CRC patients with hsa_circ_0023919 have a poor prognosis. In contrast to chemosensitive patients, chemoresistant CRC patients displayed notably reduced (A) PFS and (B) OS.

Level of Serum Hsa_circ_0023919 Serves as a Chemoresistance Diagnostic Index for CRC

To verify the prognostic efficacy of the targeted circRNA in serum, the AUC-ROC was estimated and found to be 0.8977 (95% CI: 0.8303–0.9651, Figure 4, p ≤ 0.0001). This value is consistent with its potential use as a physiological marker to distinguish between chemosensitivity and chemoresistance.

Figure 4 Levels of serum hsa_circ_0023919 serve as a diagnostic indicator for chemoresistance in CRC. To differentiate chemoresistant CRC patients before and following treatment, receiver operating characteristic curves were implemented.

Discussion

Chemotherapy remains the primary therapeutic approach for CRC, despite the limitations caused by drug resistance.23 Thus, there is an urgent need to find effective chemotherapeutic biomarkers, such as the importance of microsatellite instability (MSI) and mismatch repair (MMR) detection in predicting CRC patients receiving immunotherapy.24 There is growing evidence indicating that exosomes, which are secreted by various cell types (such as cancer cells), are released into body fluids such as blood, urine, saliva, etc.25 The diverse functions of these exosomes can be attributed to the protein (such as tetraspanins, heat shock proteins, lipid raft proteins, etc), lipid, DNA, and RNA.26,27 Furthermore, exosomal RNAs have been involved in tumorigenesis and advancement, epithelial-mesenchymal transition, blood vessel formation, immune response, and therapeutic resistance. Moreover, exosomes transported via bodily secretions may serve as clinical diagnostic biomarkers.28 The gene symbol of hsa_circ_0023919 is PICALM, and PICALM is reported to act as a pro-oncogene in CRC.20 Additionally, in response to a report, serum hsa_circ_0023919 exhibited potential as a blood biomarker in amyotrophic lateral sclerosis.21 Thus, hsa_circ_0023919 was selected as the prognostic indicator for FP+OX+BEV therapy.

The significance of circRNA activity in the development of CRC and drug resistance has gradually attracted scientific interest. For instance, hsa_circRNA_102051 inhibits the growth and metastasis of CRC by activating the Notch pathway.29 In CRC, the interaction between CircCAPRIN1 and STAT2 promotes tumor progression and lipid synthesis by increasing ACC1 expression.30 Exosomal circTUBGCP4 enhances CRC metastasis and vascular endothelial cell tipping via triggering the Akt signaling pathway.31 These findings demonstrated the significance of circRNAs in CRC drug resistance. In addition, oxaliplatin resistance in CRC is induced by a novel protein encoded by exosomal circATG4B via an enhanced autophagy process.32 In the regulation of the axis, the chemosensitivity of CRC cells to 5-fluorouracil was increased when circ_0004585 was knocked down.33 Mechanistically, many circRNA can sponge miRNA to regulate mRNA expression, thereby regulating chemotherapy resistance in CRC. For example, circ-CCS regulates oxaliplatin resistance via targeting miR-874-3p/HK2 axis in CRC.34 Circ-CD44 regulates chemotherapy resistance by targeting the miR-330-5p/ABCC1 axis in CRC.35

The efficacy and PFS from FP+OX+BEV chemotherapy were substantially correlated with plasma hsa_circ_0023919 expressions before treatment. Patients who exhibited a favorable response to FP+OX+BEV chemotherapy also had plasma hsa_circ_0023919 expression levels that were correlated with chemoresistance. Recently, several studies have demonstrated a connection between circRNA and chemoresistance in cancer. These studies have also examined circRNA in plasma and serum samples, along with cancer tissues.13,36,37 The circRNA is widely recognized for its stability in blood. The circRNAs in the serum or plasma are more feasible to quantify in clinical settings and can be monitored regularly. In relation to chemotherapy resistance, plasma hsa_circ_0023919 was collected from the same patient at various time intervals spanning from before treatment initiation to disease progression or recurrence. This finding implies that plasma hsa_circ_0023919 may serve as a viable molecular indicator for resistance measuring. This was the first investigation that presented plasma circRNA as an FP+OX+BV chemotherapy biomarker.

In conclusion, our findings suggested that the hsa_circ_0023919 expressions in plasma have the potential to serve as a prognostic indicator for assessing the efficiency as a biomarker for chemo-resistance in CRC patients who undergo treatment with the FP+OX+BEV regimen. The overall findings of this study indicated that hsa_circ_0023919 might have a role in the acquisition of resistance, thereby possibly affecting the progress of novel therapeutics for CRC.

Disclosure

The authors declare that they have no competing interests.

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