Serum Hsa_circ_0005962 Is A Prognostic Biomarker of Paclitaxel Resistance in Nonsmall Cell Lung Cancer Treatment

Background Tumor progression and the therapeutic resistance associated with cancer agents are thought to be modulated by circular RNAs (circRNAs); however, its mechanism associated with nonsmall cell lung cancer (NSCLC) is still undetermined. The following investigation aimed to evaluate the involvement of circRNAs with NSCLC. Methods The serum specimens of 146 NSCLC individuals who received complete four cycles of PTX chemotherapy were collected. The serum concentration of hsa_circ_0005962 of these individuals was assessed with quantitative real-time polymerase chain reaction (qRT-PCR), followed by the evaluation of demographic and survival consequences for further assessments. Results It was revealed that hsa_circ_0005962 is substantially increased in NSCLC chemoresistant patients and was positively correlated with the disease stage. Furthermore, the hsa_circ_0005962 value of the area under the curve was moderate, and increased hsa_circ_0005962 expression was linked with shorter overall survival (OS). Hsa_circ_0005962 stimulated paclitaxel resistance (PTX-R) in resistant NSCLC cells by regulating the axis of miR-126-5p/insulin-like growth factor 1 (IGF1). Conclusion The results of this investigation highlight that hsa_circ_0005962 induces chemoresistance in NSCLC patients and, therefore, can act as a physiological target to treat NSCLC.


Introduction
Of all the diferent types of cancers, that of the lung is frequently occurring malignant tumors and its type; nonsmall cell lung cancer (NSCLC) is believed to account for 80% to 85% of lung cancers [1,2].Although the surgical strategies, imaging technologies, and radio-and chemotherapeutics methods have developed signifcantly, the treatment efciency associated with NSCLC is still unsatisfactory.Tis is because most NSCLC cases are diagnosed only at the terminal stage due to diferent reasons [3,4].Currently, for treating terminal-stage cancer, chemotherapy is mainly applied.However, drug resistance is the principal reason behind unsuccessful chemotherapy.Effective drug development and novel strategies that could prevent or determine drug-resistant cancers can efectively improve chemotherapy efciency [5,6].
In this investigation, the circular RNA hsa_circ_0005962 locates at chr8:101936182-101937267, and its associatedgene symbol is YWHAZ.Te serum concentration of hsa_circ_0005962 in NSCLC individuals and its correlation with clinical outcomes were determined.It was revealed that PTX-R NSCLC individuals had increased hsa_circ_0005962 expressions, which were associated with substandard overall survival (OS).Hsa_circ_0005962 induced NSCLC individuals PTX resistance by regulating miR-126-5p/IGF1 axis.Furthermore, hsa_circ_0005962 presented a reasonable area under the receiver operating characteristic (AUC-ROC) curve data, indicating its importance as a new prognostic bio-index of PTX-R NSCLC.

Materials and Method
2.1.Clinical Samples and Cell Culture.Serum specimens of 146 NSCLC and 142 healthy control individuals enrolled at First People's Hospital of Jiujiang were obtained.Tis investigation was authenticated by the ethical board of the First People's Hospital of Jiujiang, and all the subjects were initially informed about the research, and then, their signed consent was taken.Primary human bronchial epithelial (HBE) and NSCLC (A549 and H460) cells were acquired from Procell (Wuhan, China).Te PTX-R corresponding NSCLC cells (H460-PTX and A549-PTX) was developed by augmenting the parental cells with accelerating PTX concentrations (SP8020; Solarbio, Beijing, China).All the cultures were propagated in RPMI1640 media (Invitrogen, Carlsbad, CA, USA) augmented with 10% fetal bovine serum (FBS; Invitrogen) and 1% penicillin-streptomycin (Invitrogen) at the standard temperature of 37 °C and CO 2 percentage of 5. Additionally, 5 nM of PTX (Solarbio) was introduced in the growth media to conserve H460-PTX and A549-PTX cell resistance.

Statistical Evaluation.
All the protocols were performed thrice, and their data were assessed with GraphPad Prism 7 and depicted by values of the mean of ± standard deviation.Student's t-test and one-way analysis of variance (ANOVA) were carried out for diferential analysis.P value <00.5 was termed signifcant.

NSCLC Patients Exhibit Upregulation of Serum
Hsa_circ_0005962.To assess the efciency of hsa_-circ_0005962 as a physiological chemoresistance index, its serum concentration was assessed.NSCLC individuals showed notably increased serum hsa_circ_0005962 expression than control individuals (Figure 1(a)).Moreover, its expression was greater in PTX-R (n � 64) than in PTXsensitive patients (n � 82) (Figure 1(b)).Te data indicated the efcacy of this circRNA as an index for NSCLC chemotherapy.In addition, resistance to digestion with RNase R exonuclease specifcally degraded linear RNAs but not circRNAs (Figure 1(c)).Te results of actinomycin D assays revealed that the half-life of the hsa_circ_0005962 transcript exceeded 24 h, longer than the half-life of YWHAZ mRNA, indicating that hsa_circ_0005962 is more stable than the linear YWHAZ transcript (Figure 1(d)).

Hsa_circ_0005962 Expressions Are Related to NSCLC
Patients' Clinical Manifestations.Next, the NSCLC individuals were stratifed into groups with high and low hsa_circ_0005962 concentrations in serum, according to their average expression in this cohort.Furthermore, the clinical manifestations were also compared between the two cohorts.Chi-squared analyses indicated the association of hsa_-circ_0005962 expressions with tumor size, TNM stages, distant metastasis or recurrence, and lymph node metastasis (Table 1); however, it was negatively related to age, gender, or tumor type.Kaplan-Meier assessment suggested that individuals who exhibit enhanced hsa_circ_0005962 expression have shorter OS than those with low expression levels (Figure 2).International Journal of Clinical Practice individuals (Figure 3).Cox proportional hazards regression analyses indicated the association of hsa_-circ_0005962 levels with tumor size, TNM stages, distant metastasis or recurrence, lymph node metastasis, and chemoresistance of patient PFS (Table 2) and OS (Table 3), emphasizing the importance of this circRNA as an independent predictor of survivability of chemoresistant NSCLC patients.

Serum hsa_circ_0005962 Concentrations Are a Diagnostic
Index for NSCLC Chemoresistance.To determine the diagnostic efciency of concerned circRNA in serum, the AUC-ROC was assessed and observed to be 0.9014 (95% CI, 0.8661-0.9367, Figure 4, p < 0.0001), compatible with its application as a physiological marker for differentiating NSCLC sufering individuals from healthy subjects.

Discussion
Most of the chemotherapeutic agents fail to successfully treat cancer because of chemoresistance in humans, including resistance against NSCLC.Advancements in highly efcient sequencing technology have allowed the discovery of diverse circRNAs that regulate chemoresistance development.NSCLC individuals' serum indicated substantially increased hsa_circ_0005962 levels relative to the control cohort.Furthermore, hsa_circ_0005962 upregulation in PTX-R NSCLC individuals relative to chemosensitive individuals  4 International Journal of Clinical Practice indicated its importance as an independent predictor of clinical outcomes.In addition, hsa_circ_0005962 stimulated PTX-R by controlling miR-126-5p/IGF1 axis.Te importance of circRNAs activity in NSCLC development and drug resistance has slowly gained a lot of attention from scientists.For example, the circ-CPA4/let-7 miRNA/PD-L1 axis modulates the propagation, stemness, immune evasion, and drug resistance of NSCLC cells [17].Suppressing circ_0014130 inhibits resistance against drugs and the malignant function of docetaxel-resistant NSCLC cells by controlling the axis of miR-545-3p-yes-associated protein 1 (YAP1) [18].Te hsa_circ_0002874 overexpression induces PTX resistance in NSCLC by controlling miR-1273f/murine double minute 2 (MDM2)/p53 pathway [19].Tese studies indicate the important function of circRNAs in drug resistance in NSCLC.Additionally, circRNAs are also excellent physiological markers such as circ_0001649 acts as a marker by inhibiting NSCLC progression by miR-331-3p and miR-338-5p sponging [20], hsa_circ_0033155 is also a novel NSCLC index [21], and hsa_circ_0102533 is a blood-based marker for diagnosing NSCLC and regulating in vitro apoptosis [22].
In this investigation, the hsa_circ_0005962 expressions were elevated in both PTX-R and PTX-sensitive NSCLC individuals, indicating its importance as a valuable predictor of chemotherapeutic responses.Hsa_circ_0005962

Conclusion
Tis investigation summarizes that the upregulation of hsa_circ_0005962 is prominently evident in the NSCLC individuals' serum samples, where this upregulation was substantially pronounced in the serum of chemosensitive rather than chemoresistant subjects.Furthermore, hsa_-circ_0005962 promoted PTX resistance in PTX-R NSCLC cells by modulating miR-126-5p/IGF1 axis.Terefore, hsa_circ_0005962 is an efective target that opens a new path for further studies to evaluate the underlying processes involved in NSCLC chemoresistance.

Figure 1 :
Figure 1: NSCLC patients exhibit serum hsa_circ_0005962 upregulation.(a) Serum hsa_circ_0005962 levels were higher in NSCLC patients.(b) PTX-resistant patients exhibited higher levels of hsa_circ_0005962 expression compared with PTX-sensitive patients in the before and after treatments (n � 80).(c) Te expression of hsa_circ_0005962 and YWHAZ mRNA after treatment with RNase R in A549/ PTX and H460/PTX cells.(d) Te expression of hsa_circ_0005962 and YWHAZ mRNA after treatment with actinomycin D at the indicated time points in A549/PTX and H460/PTX cells.* p < 0.05.

Figure 2 :
Figure 2: Hsa_circ_0005962 levels are related to clinical features in NSCLC patients.Patients exhibiting higher hsa_circ_0005962 expression levels exhibited prolonged overall survival compared with patients with lower levels.

Table 1 :
Correlations between hsa_circ_0005962 levels and NSCLC patient clinicopathological features.

Table 2 :
Univariate and multivariate analyses of NSCLC patient progression-free survival.

Table 3 :
Univariate and multivariate analyses of NSCLC patients' overall survival.

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International Journal of Clinical Practice concentrations were associated with tumor size, TNM stages, distant metastasis or recurrence, and lymph node metastasis in NSCLC individuals and were not related to age, gender, and tumor type.Kaplan-Meier method suggested that increased hsa_circ_0005962 expressions were linked with short OS compared with low levels.Furthermore, chemoresistant subjects had shorter OS and PFS than chemosensitive subjects.Univariate and multivariate analyses suggested that tumor size, TNM stages, distant metastasis or recurrence, lymph node metastasis, and chemoresistance were correlated with PFS and OS, indicating that hsa_circ_0005962 is an efective independent outcome predictor for NSCLC individuals.Additionally, the AUC value (0.9014) indicated that serum expression of hsa_circ_0005962 can be reliably utilized for diferentiating NSCLC individuals from healthy subjects.