circ_ZFR Is Linked to Paclitaxel Resistance in Cervical Cancer via miR-944 Sponging and IL-10 Upregulation

Objective Cervical cancer (CC) has an elevated rate of invasion and death despite surgical treatment, radiotherapy, and chemotherapy. Several studies revealed that circRNAs have a key contribution to the resistance of drugs against different types of carcinomas. The goal of the existing study was to figure out what role circ_ZFR plays in paclitaxel (PTX) resistance in cervical cancer (CC) patients. Materials and Methods Herein, two types of CC cells (SiHa/PTX and Hela/PTX) were utilized. The levels of IL-10 mRNA, miR-944, and circ_ZFR were measured using qRT-PCR analyses. The CCK-8 assay was used to determine PTX resistance. The IL-10 expression was measured via the ELISA technique. The combination of miR-944 and circ_ZFR or IL-10 was validated using a dual-luciferase reporter (DLR) assay. Results The amount of circ_ZFR was increased in PTX-resistant CC cells and tissues. In PTX-resistant CC cells, knocking down circ_ZFR expression decreased PTX resistance. circ_ZFR knockdown significantly reduced IL-10 expression via sponging miR-944, increasing PTX sensitivity in PTX-resistant CC cells. Conclusion circ_ZFR knockdown has a considerable role in overwhelming CC-associated PTX resistance by modifying the axis of miR-944/IL-10 axis, suggesting that developing a circRNA target-based treatment could be considered prevent CC progression.


Introduction
According to the global survey of cancer statistics, cervical cancer (CC) is considered to be the fourth most prevalent malignancy in women despite the HPV (human papillomavirus) vaccination and testing [1,2]. CC has an elevated rate of invasion and death despite surgical treatment, radiotherapy, and chemotherapy. For resistant or recurring CC, the antitumor drug paclitaxel (PTX) is now commonly utilized, but PTX resistance has a significant impact on its therapeutic benefit [3]. As a result, enhancing clinical treatment methods requires extensive knowledge of the molecular mechanisms underlying PTX resistance in CC.
The critical role of the circRNA-miRNA-mRNA regulation network in chemoresistance has been gradually explored [19][20][21]. According to our obtained data, miR-944 has the same binding sequences as circ_ZFR and IL-10 in the existing study, suggesting that circ_ZFR could act as a sponge for miR-944 to boost IL-10 expression. As a result, we decided to evaluate the implications of the circ_ZFR/ miR-944/IL-10 axis for CC PTX resistance.

Materials and Methods
2.1. Tissue Acquisition. At Wenling First People's Hospital, 96 CC tissue samples (62 cervical squamous cell carcinoma and 34 cervical adenocarcinoma) and surrounding healthy tissue specimens were taken from patients associated with CC, followed by classification into two groups, i.e., the treatment-resistant group (n = 50) and the treatmentsensitive (n = 46) group. The existing study was authorized by the Wenling First People's Hospital Ethics Committee. All of the patients who took part in this study signed a written informed consent form.
2.5. Dual-Luciferase Reporter (DLR) Assay. The complete or mutant segments of the circ_ZFR or IL-10 3 ′ UTR comprising the expected miR-944 interacting sites were cloned downstream of the luciferase gene in the pGL3 vector (Pro-mega, Fitchburg, WI, USA). The cotransfection of 293 T cells was carried out with miR-NC or miR-944 in combination with IL-10 3′UTR-MUT or IL-10 3′UTR-WT or circ_ ZFR-MUT or circ_ZFR-WT. After 24 hrs of the incubation period, luciferase activity was assessed using the DLR assay.
2.6. RNA Immunoprecipitation (RIP) Assay. The RIP assay was carried out with the help of the Magna RIP kit (Millipore). On the basis provided instructions (user's manual), Hela/PTX and SiHa/PTX cell transfection was carried out with miR-944 or miR-NC and cultured for 48 hrs before being lysed with RIP buffer having IgG-antibody or Ago2-labeled magnetic beads. Following that, TRIzol reagent was utilized to obtain immunoprecipitated RNAs. Furthermore, the circ_ZFR enrichment was determined via qRT-PCR evaluations.
2.7. RNA Pull-Down Assay. SiHa/PTX and Hela/PTX cell transfection was carried out via biotinylated circ_ZFR-MUT or ZFR-WT or NC, followed by 48 hrs of incubation. The cells were collected, lysed with RIP buffer, and then treated with streptavidin magnetic beads (M-280; Invitrogen) according to the manufacturer's instructions. The beads were thricely rinsed via a cold buffer after being incubated in a cold chamber (4°C) for two hours. Eventually, the RNA elution was carried out so that it could be used in other experimental evaluations.
2.8. ELISA. Following the instructions of the provider, an ELISA kit (R & D Systems) was employed for the measurement of IL-10 expression in various culture media. The average value was calculated from three experiments evaluated separately.
2.9. Statistics Analysis. The obtained data was compiled using SD from triplicated expressions (individually performed). GraphPad Ver 7.04 (La Jolla, CA, USA) was employed for the statistical evaluations on the data obtained. The means of continuous outcome variables were examined via ANOVA. Kaplan-Meier plots and log-rank test were conducted for the analysis of survival rates and to investigate variations among subgroups of patients. Variations were regarded as statistically considerable when the p values were less than 0.05.

An Elevated Expression of circ_ZFR in CC Tissues and
Cell Lines (Resistant to PTX). The circ_ZFR expression was evaluated in PTX-sensitive and PTX-resistant CC tissues via qRT-PCR analysis. The obtained data suggested that circ_ZFR expression was found to be elevated in CC tissues (resistant to PTX) in comparison with the healthy tissues and CC tissues (sensitive to PTX), as represented in Figure 1(a). Patients who were PTX-sensitive had a significant survival rate when treated with PTX, as indicated in Figure 1(b). The results of ROC curves showed that circ_ ZFR may have diagnostic significance for PTX-resistant CC patients (Figure 1(c)). Furthermore, in HeLa and SiHa cells, the level of circ_ZFR was decreased relative to HeLa/ PTX and SiHa/PTX cells, as depicted in Figure 1(d). The results obtained from the CCK-8 assay showed PTX resistance in HeLa/PTX and SiHa/PTX cells, as revealed by the higher IC 50 concentration of PTX in HeLa/PTX and SiHa/ PTX cells, as depicted in Figure 1(e). circ_ZFR was mostly found in the cytoplasmic part of the underlined cells, as indicated in Figure 1(f). The underlined data revealed that dysregulated circ_ZFR may have a role in the chemoresistance of CC.

Suppression of PTX Resistance in CC Cells (Resistant to PTX) by circ_ZFR Knockdown.
To determine the role of circ_ZFR in PTX-resistant CC cells, si-circ_ZFR was transfected into HeLa/PTX and SiHa/PTX cells to knock down the circ_ZFR expression, and relative to si-NC groups, a considerable decrease was observed in the expression of circ_ZFR in HeLa/PTX and SiHa/PTX cells, as suggested by the qRT-PCR (Figure 2(a)). PTX IC 50 value was lowered in the underlined cells (transfected with si-circ_ ZFR), as indicated by CCK-8 assay which suggested that circ_ZFR knockdown overcomes PTX resistance in the underlined cells, as depicted in Figure 2(b). In PTXresistant CC cells, knocking down circ_ZFR boosted the sensitivity of PTX.

Attenuation of miR-944
Enhanced the Impact of circ_ ZFR Knockdown on PTX Sensitivity in CC Cells (Resistant to PTX). Online tool starbase 3.0 was employed to examine the possible target of circ_ZFR to explore the pathway associated with circ_ZFR to regulate PTX resistance and cellular progression in CC cells (PTX resistant). miR-195-5p could be a circ_ZFR target, as it had the binding sites of circ_ ZFR (Figure 3(a)). The DLR assay revealed that miR-994 transfection significantly reduced circ_ZFR-WT luciferase activity while not affecting circ_ZFR-MUT luciferase activity in 293 T cells, validating the link between circ_ZFR and miR-944, as depicted in Figure 3(b). Anti-AGO2 antibody was used to enrich endogenous circ_ZFR (Figure 3(c)). In HeLa/PTX and SiHa/PTX cells, the wild-type circ_ZFR collected more miR-944 than the mutant revealed by RNA pulldown assay (Figure 3(d)), showing that circ_ZFR directly targets miR-944. Furthermore, we discovered that silencing circ_ZFR increased the expression of miR-944 in the underlined cells, as depicted in Figure 3(e). The expression of miR-944 was elevated in HeLa and SiHa cells relative to HeLa/ PTX and SiHa/PTX cells, as depicted in Figure 3(f). Furthermore, the miR-944 level decreased from normal tissues to CC tissues (PTX-sensitive) and then to CC tissues (PTXresistant), as indicated in Figure 3(g). It has also been depicted that there was a negative association, existed between miR-944 and the circ_ZFR expression in tumor tissues (Figure 3(h)). miR-944 was negatively regulated by circ_ZFR through directly targeting. Based on the above findings, rescue experiments were conducted to evaluate whether PTX resistance has been regulated by circ_ZFR through miR-944 targeting. An elevated expression of miR-944 (inducted by si-circ_ZFR) was found to be reversed via anti-miR-944 transfection in HeLa/PTX and SiHa/PTX cells, as indicated in Figure 3(i). By lowering miR-944, the inhibitory effects of circ_ZFR knockdown on PTX resistance were all reversed in HeLa/PTX and SiHa/PTX cells (Figure 3(j)).
The underlined results indicated that through sponging miR-944, circ_ZFR knockdown suppressed PTX-resistant CC cells.   Figure 4(a). Then, using a DLR assay, it was discovered that in miR-944 and IL-10-WT cotransfected 293 T cells; luciferase activity was decreased in comparison to miR-NC and IL-10-WT cotransfected groups, but variations were not observed in IL-10-MUT groups (Figure 4(b)). To determine the impact of miR-944 on the expression of IL-10, miR-944, and anti-miR-944, their controls were transfected. HeLa/PTX and SiHa/ PTX cells were successfully transfected with miR-944 and anti-miR-944, as shown in Figure 4(c). Furthermore, we evaluated that elevated expression of miR-944 considerably lowered the expression of IL-10 in HeLa/PTX and SiHa/PTX cells but attenuation of miR-944 showed apposing results (Figures 4(d) and 4(e)). Furthermore, our findings revealed that IL-10 expression was decreased in HeLa and SiHa cells relative to HeLa/PTX and SiHa/PTX cells, as depicted in Figures 4(f) and 4(g). From healthy tissues to tumor tissues (PTX-sensitive) and subsequently to tumor tissues (PTX-resistant), the expression of IL-10 was elevated (Figure 4(h)). The level of IL-10 mRNA in CC tissues was inversely linked with miR-944 expression using Spearman's correlation coefficient analysis, as revealed in Figure 4(i). miR-195-5p transfection significantly reduced IL-10 expression in HeLa/PTX and SiHa/PTX cells, as depicted in Figures 4(j) and 4(k), whereas the effects were restored by IL-10 transfection. miR-944 elevated expression successfully decreased PTX resistance in HeLa/PTX and SiHa/PTX cells using the CCK-8 assay, while elevated IL-10 successfully reversed the results (Figure 4(l)). In other words, elevated expression of miR-944, which targets IL-10, plays a considerable role in PTX sensitivity in CC cells (PTX-resistant).

Discussion
Chemoresistance is a major obstacle in the treatment of human malignancies, including CC. Various circRNAs have been confirmed to have a role in regulating the development of chemoresistance [22,23]. The existing study is aimed at seeing what role circ_ZFR plays in modulating CC's PTX sensitivity. As a result of the modulation of the miR-944/ IL-10 axis by circ_ZFR, PTX resistance in CC cells (PTXresistant) was facilitated. The importance of circRNAs in tumor growth and treatment resistance has steadily drawn researchers' attention in CC. circMTO1, for example, promotes cervical cancer growth and chemoresistance by miR-6893 regulation [21]. hsa-circ 0023404 sponging miR-5047 elevated progression and chemoresistance in cervical cancer via VEGFA and autophagy signaling [24]. The underlined findings revealed that circRNAs performed a variety of roles in CC drug resistance. circ_ZFR has a role in the paclitaxel resistance and development of NSCLC by elevated expression of KPNA4 through sponging miR-195-5p [25]. By modulating the miR-545-3p/CBLL1 axis, circ 0072083 interference suppressed DDP resistance, formation of cell colony, cell cycle, and metastasis and accelerated the apoptotic process in NSCLC cells [26]. In this study, a high level of circ_ZFR was found in chemoresistant CC tissues and cells. In vitro, circ_ZFR deficiency increased PTX sensitivity in CC cells (resistant to PTX). circ_ZFR has a significant contribution to PTX resistance in CC as a whole. circ_ZFR was evaluated as the sponge for miR-944, which then boosted IL-6 expression, according to the mechanistic analysis. The miR-944/FZD7 axis attenuated doxorubicin resistance and reduced tumor growth in colorectal cancer when the circular RNA circCSPP1 was knocked down [27]. The obtained results revealed that miR-944 appeared to have a considerable contribution to the development and chemoresistance of CC. Our findings suggested that miR-944 levels were lowered in CC tissues and cells (resistant to PTX). The impact of circ_ ZFR knockdown on PTX sensitivity may be effectively restored by suppressing miR-944 in CC cells (PTX-resistant). Furthermore, elevated expression of miR-944 boosted the sensitivity of PTX in cells (PTX-resistant); however, these effects might be reversed by an elevated expression of IL-10. Based on the obtained results, miR-944 decreased PTX resistance in CC cells (PTX-resistant) by targeting IL-10. In support of our results, AS101 sensitizes tumors to chemotherapy by blocking the tumor IL-10 autocrine loop which supports our findings [28]. Although IL-10 is a target of a variety of miRNAs, including miR-374b-5p [29] and miR-98 [30], we are the first to show an interaction between miR-944 and IL-10 in CC chemoresistance.
In conclusion, the obtained data from the existing study revealed that circ_ZFR enhanced PTX resistance in CC by modulating the miR-944/IL-10 axis, which may have a candidate therapeutic strategy to decrease the resistance of CC to PTX.