Serum hsa_circ_0000615 is a prognostic biomarker of sorafenib resistance in hepatocellular carcinoma

Abstract Background Circular RNAs (circRNAs) can shape tumor progression and chemoresistance. How specific circRNAs shape hepatocellular carcinoma (HCC) chemoresistance, however, remains to be fully elucidated. Methods In total, serum samples were collected from 202 HCC patients that had completed four sorafenib chemotherapy cycles. Serum hsa_circ_0000615 levels in these patients were quantified via quantitative real‐time polymerase chain reaction (qRT‐PCR), with demographic details and survival outcomes being recorded for subsequent analyses. Results We found hsa_circ_0000615 to be significantly upregulated in chemoresistant HCC patients relative to chemosensitive patients, with such upregulation being positively correlated with disease stage. Moreover, the area under the curve (AUC) value for hsa_circ_0000615 was moderately good, and high levels of hsa_circ_0000615 expression were associated with shorter overall survival among chemoresistant HCC patients. Conclusion Our results highlight hsa_circ_0000615 as a promising driver of sorafenib resistance in HCC patients, highlighting it as a promising target for the treatment of this deadly cancer type.

gastric, 16 and colorectal cancers. 17 Moreover, hsa_circ_0000615 has been found to promote HCC cell migration, invasion, stemness, and proliferation. 18,19 How hsa_circ_0000615 functions in the context of tumor chemoresistance, however, remains to be defined.
Herein, we explored the expression of hsa_circ_0000615 in HCC patient serum and its relationship with patient clinical findings.
Overall, we found that sorafenib-resistant HCC patients exhibited hsa_circ_0000615 upregulation that was related to poorer overall survival (OS) outcomes. Moreover, hsa_circ_0000615 exhibited reasonably good area under the ROC curve (AUC) values, suggesting that it may offer value as a novel prognostic biomarker of sorafenibresistant HCC.

| Statistical analysis
Data are means ± standard deviation (SD) and were compared via Student's t tests using GraphPad Prism 7. The Kaplan-Meier method was used for survival analyses, with p < .05 as the threshold of significance.

| Hepatocellular carcinoma patients exhibit serum hsa_circ_0000615 upregulation
We began by assessing the levels of hsa_circ_0000615 in control  Figure 1B). Moreover, hsa_circ_0000615 expression levels were higher in sorafenib-resistant patients (n = 122) relative to those in sorafenib-sensitive individuals (n = 80) ( Figure 1C). As such, hsa_circ_0000615 offers potential value as an HCC chemotherapy biomarker.
F I G U R E 1 Hepatocellular carcinoma (HCC) patient serum samples exhibit hsa_circ_0000615 upregulation. (A). Hsa_circ_0000615 levels were significantly increased in sorafenib-resistant HCC cells. (B). Serum hsa_circ_0000615 levels were higher in HCC patients. (C). sorafenibresistant patients (n = 122) exhibited higher levels of hsa_circ_0000615 expression compared with sorafenib-sensitive patients in the before treatment and after treatment (n = 80). *p < .05.

| Hsa_circ_0000615 levels are related to clinical features in HCC patients
Next, we stratified HCC patients into those with high and low levels of serum hsa_circ_0000615 based on the mean expression of this circRNA in this cohort and compared clinical features between these two patient groups. Chi-squared analyses revealed that hsa_ circ_0000615 expression was associated with clinical stage, TNM stage, and lymph node metastasis (Table 1), but was unrelated to age, sex, or histological grade. Kaplan-Meier analyses indicated that patients exhibiting higher levels of hsa_circ_0000615 expression presented with shorter OS relative to patients expressing low hsa_circ_0000615 levels ( Figure 2).

| Hsa_circ_0000615 is associated with poor chemoresistant hepatocellular carcinoma patient prognosis
Through Kaplan-Meier analyses and log-rank tests, we found chemoresistant HCC patients to exhibit significantly reduced OS and progression-free survival (PFS) compared with chemosensitive patients (Figure 3). Through Cox proportional hazards regression analyses, we determined that clinical stage, chemoresistance, TNM stage, lymph node metastasis, and hsa_circ_0000615 levels were associated with patient PFS (Table 2) and OS (Table 3), highlighting hsa_circ_0000615 as a promising independent predictor of chemoresistant HCC patient survival.

| Serum hsa_circ_0000615 levels offer diagnostic utility for the detection of hepatocellular carcinoma chemoresistance
Previous studies have shown that circRNAs show excellent potential diagnostic utility in various cancers, such as, breast cancer, 20 gastric cancer, 21 and HCC. 22 To assess the potential diagnostic utility of serum hsa_circ_0000615 in patients with HCC, the area under the receiver operating characteristic (ROC) curve (AUC) was determined and found to be 0.9238 (95% CI, 0.8915-0.956, Figure 4,

| DISCUSS ION
Herein, we found that serum samples from HCC patients exhibited significant increases in hsa_circ_0000615 levels compared with those from control individuals. Moreover, the upregulation of this circRNA in samples from sorafenib-resistant HCC patients relative to those from chemosensitive patients suggested that it may offer value as an independent predictor of patient outcomes.
A growing body of evidence suggests that circRNAs are functionally important in cancer and may offer value as predictive biomarkers or therapeutic targets. In colorectal cancer, for example, circRNA_0000392 can promote tumor progression via the miR-193a-5p/PIK3R3/AKT axis. 23 Moreover, in non-small cell lung cancer, circNDUFB2 can destabilize IGF2BPs and activate antitumor immune responses to suppress tumor progression. 24 In HCC, circRNA-SORE can stabilize YBX1 to drive sorafenib resistance. 25 As such, circRNAs function in a tumor-specific manner.
Neoadjuvant chemotherapy is a mainstay of treatment for many cancers and has been used with increasing frequency over the past decade, with sorafenib-based neoadjuvant chemotherapy being a standard of care for HCC patients. Those HCC patients that undergo sorafenib-based chemotherapy prior to radical cystectomy exhibit better OS outcomes, but a subset of patients fail to attain any benefit from such treatment, with pathological responses to neoadjuvant chemotherapy being predictive of disease-specific survival outcomes. Identifying reliable biomarkers capable of guiding clinicians to the selection of patients most likely to benefit from chemotherapeutic intervention is thus a critical clinical task.
In HCC, circRNAs can function as central regulators of sorafenibresistance, 26-28 with hsa_circ_0000615 having previously been shown to drive HCC tumor growth and metastatic progression. 18,19 In this study, we further found hsa_circ_0000615 to be expressed at significantly higher levels in Hep G2/sorafenib and Huh 7/sorafenib cells relative to corresponding parental cell lines.

| CON CLUS IONS
In summary, we herein found hsa_circ_0000615 upregulation to be prominent within serum samples from HCC patients, with such upregulation being significantly more pronounced in samples from chemosensitive patients relative to chemoresistant patients. As such, hsa_circ_0000615 is a promising target that warrants further study in an effort to understand the mechanistic basis for HCC patient chemoresistance.

ACK N OWLED G M ENTS
This study was supported by the Key Project of Anhui Provincial Department of Education (KJ2021A0799).

CO N FLI C T O F I NTE R E S T
The authors of this work declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Due to the nature of this research, participants of this study did not agree for their data to be shared publicly, so supporting data are not available.

O RCI D
Xiaolin Ding https://orcid.org/0000-0001-6285-769X F I G U R E 4 Serum hsa_circ_0000615 levels offer diagnostic value as predictors of hepatocellular carcinoma (HCC) patient chemoresistance. Receiver-operating characteristic curves were used to differentiate between chemoresistant HCC patients before and after therapy.