Circular RNA-AnnexinA7 accelerates cisplatin resistance in non-small cell lung cancer via modulating microRNA-545-3p to mediate Cyclin D1

Objective: To explore the mechanism of circular RNA (circRNA)-AnnexinA7 (ANXA7) in non-small cell lung cancer (NSCLC) cisplatin (DDP) resistance through microRNA (miR)-545-3p to target Cyclin D1 (CCND1). Meth-ods: DDP-resistant and non-resistant NSCLC tissues and normal tissues were collected. DDP-resistant cells (A549/ DDP and H460/DDP) were constructed. circ-ANXA7, miR-545-3p, CCND1, P-Glycoprotein, and glutathione S-transferase-π in tissues and cells were measured. Analysis of circ-ANXA7 ring structure was performed, as well as detection of circ-ANXA7 distribution in cells. Cell proliferation was detected by MTT and colony formation assay, apoptosis rate was detected by flow cytometry, and cell migration and invasion were evaluated by Transwell assay. The targeting relationship between circ-ANXA7, miR-545-3p and CCND1 was verified. Measurement of tumor volume and quality in mice was performed. Results: Circ-ANXA7 and CCND1 were elevated, while miR-545-3p was suppressed in DDP-resistant NSCLC tissues and cells. Circ-ANXA7 combined with miR-545-3p, which targeted CCND1 to expedite A549/DDP cell proliferation, migration, invasion, DDP resistance, but inhibited cell apoptosis. Conclusion: Circ-ANXA7 enhances DDP resistance in NSCLC via absorbing miR-545-3p to target CCND1 and might be a latent therapeutic target for NSCLC.


INTRODUCTION
Lung cancer (LC) is a malignant tumor with the uppermost morbidity and mortality in the world (Zhang et al., 2021).Non-small cell lung cancer (NSCLC) is an extremely critical type of LC, taking up about 85% of LC cases (Xu et al., 2020).Treatment strategies for NSCLC have improved recently, but the 5-year survival rate is still greatly reduced by about 10-15% (Xu et al., 2021).Cisplatin (DDP) chemotherapy is a first-line anti-cancer chemotherapy agent in NSCLC.Nevertheless, patients treated with DDP for a long time develop DDP resistance (Wu et al., 2020).As reported, around 63% of NSCLC patients have DDP resistance (Ye et al., 2020).Consequently, lessening DDP resistance is the crux to ameliorating NSCLC patients' outcomes.This study was to comprehend latent mechanisms in NSCLC DDP resistance and identify latent biomarkers, offering brandnew insights into NSCLC therapy.
Circular RNAs (circRNAs), a non-coding RNA with closed continuous loops, have been testified to mediate primary or antitumor responses in different cancer treatments (Fan et al., 2021).Notably, numerous circRNAs exert critical roles in multiple biological processes of NSCLC.For instance, circ_PIP5K1A (Feng et al., 2021), circ-RNF121 (Liu et al., 2022) and circ_0076305 (Wang et al., 2021) have been reported to be implicated in the cancer progression and DDP resistance of NSCLC.Circ-AnnexinA7 (ANXA7), as a member of circRNA, has been reported to expedite lung adenocarcinoma progression (Wang, 2021).In the preliminary experiment, this circRNA was aberrantly modulated in DDP-resistant NSCLC tissues.Nevertheless, the latent impact of circ-ANXA7 on DDP resistance in NSCLC is unknown.
Cyclin D1 (CCND1), a recognized cell cycle-associated protein, exerts a crucial action in cell cycle change (Meng et al., 2021).CCND1 is a critical driver of malignant transformation and is frequently elevated in NSCLC, leading to the aberrant proliferation of NSCLC cells (Liu et al., 2020).Notably, modulating CCND1 can alter DDP-resistant cell malignant phenotype (Ju et al., 2020).In this study, it was hypothesized that circ-ANXA7 might participate in NSCLC DDP resistance via miR-545-3p to modulate CCND1.
In this study, 2 DDP-resistant NSCLC cell lines (A549/DDP and H460/DDP) were constructed, and circ-ANXA7 was confirmed to modulate DDP-resistant NSCLC cell sensitivity and behavior.Additionally, this research further elucidated the action of circ-ANXA7/ miR-545-3p/CCND1 axis in DDP resistance and cancerization of NSCLC.

Clinical tissue specimen
90 tissue samples were obtained from the School of Medicine, University of Electronic Science and Technology of China, including 30 non-tumor tissue samples from patients with pulmonary laceration repair (control) and 60 NSCLC tissue samples from NSCLC patients with radical resection.Patients with progressive disease (PD) or postoperative recurrence less than 6 months were DDP-resistant, while patients without PD and postoperative recurrence over 12 months were non-resistant.All tissue samples were placed at -80°C.Authorization of the study was performed by the Ethics Committee of the School of Medicine, University of Electronic Science and Technology of China, and written informed consent was obtained from all participants.
Actinomycin D test: Cells were cultured with or without 2 µg/ml actinomycin D (Sigma, USA) and harvested at different points in time.Circ-ANXA7 and ANXA7 mRNA was determined by RT-qPCR (Wu et al., 2020).

Subcellular localization analysis
Cytoplasm RNA and nuclear RNA of cells were isolated using PARIS Kit (Invitrogen).Then, the examination of circ-ANXA7 in cytoplasm RNA and nuclear RNA was implemented by RT-qPCR.U6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were nuclear and cytoplasmic controls, respectively.

RT-qPCR
Extraction of total RNA from NSCLC tissues and cells was done using Trizol reagent (Invitrogen).Reverse transcription of circRNA/mRNA and miRNA was performed with PrimeScript RT reagent Kit and miRNA First Strand Synthesis Kit (both Takara, Tokyo, Japan), respectively.RT-qPCR was performed with SYBR Green kit (Thermo Fisher Scientific, Waltham, MA, USA) and Mx3005P qPCR system (Agilent Technologies, Santa Clara, CA, USA).U6 and GAPDH were loading controls for miRNA and mRNA/circRNA, respectively.Primer sequences were presented in Table 1 (Song et al., 2021).

Colony formation assay
A549/DDP cells (5×10 3 /well) were seeded in 12well plates and cultured for 14 d, during which a fresh complete medium was replaced every 3 days and the wells were washed twice with PBS.Then, colonies were fixed in paraformaldehyde (4%, Beyotime), dyed with 0.1% crystal violet (Beyotime) for 2 h, and washed with ddH 2 O 3 times.The number of colonies was counted under an inverted microscope (Nikon).

Transwell migration and invasion analysis
Transwell assay evaluated A549/DDP cell invasion and migration.For migration and invasion measurements, transwell chambers were used without or with Matrigel, respectively (Corning, USA).Cells were prepared at 2×10 6 cells/mL, then 400 μL cell suspension was inoculated into the upper chamber containing serum-free medium, and 10% FBS was added to the lower chamber.After 24 h of culture, cells in the upper layer were removed with cotton swabs, while those in the lower layer were fixed with methanol for 30 min, stained with 0.1% crystal violet, and counted in five fields under a microscope (Swiss taikang, magnification ×100) (Guo et al., 2022).

RNA immunoprecipitation (RIP) experiment
Anti-Ago2 (ab252812) and anti-IgG (ab109489) antibodies were utilized for detection.In short, cells were lysed with a lysis buffer and then incubated with protein-g magnetic beads-conjugated anti-Ago2 or IgG at 40°C for 6 h.Microbeads were later collected, and binding RNA was extracted to examine the enrichment of circ-ANXA7 and miR-545-3p (Zhang et al., 2021).

In vivo tumor growth test
The tumor formation experiment was conducted in BALB/c male nude mice (n=24, 6 weeks old, weight of 18-20 g, Huafukang, Beijing, China).A549/ DDP cells transfected with sh-Circ-ANXA7, or sh-NC (1×10 7 ) were injected subcutaneously into the back of mice.Mice were divided into four groups: sh-NC+PBS, sh-NC+DDP, sh-circ-ANXA7+PBS, and sh-circ-ANXA7+DDP groups.DDP (5 mg/kg) or PBS was injected to measure the tumor volume once a week.After 4 weeks, transplanted tumors from euthanized mice were weighed.Animal treatments were approved by the Animal Research Committee of the School of Medicine, University of Electronic Science and Technology of China (Shao et al., 2021).

Statistical analysis
Statistical software SPSS 21.0 (SPSS, Inc, Chicago, IL, USA) was utilized to analyze data.Kolmogorov-Smirnov test checked the normal distribution of data, and the results were presented as mean ± standard deviation (S.D.).Two-group comparisons were performed using ttest.Comparisons among multiple groups were imple-  mented with one-way analysis of variance (ANOVA) and pairwise comparison after ANOVA analysis was performed using Fisher's Least Significant Difference t-test.
Enumeration data were shown as rate or percentage and analyzed by Chi-square test.*p<0.05indicates a significant difference.

Circ-ANXA7 expression is modulated in DDP-resistant NSCLC tissues and cells
circ-ANXA7 levels in resistant and non-resistant NSCLC tissues and normal tissues were tested by RT-qPCR.Compared with non-resistant NSCLC tissues and normal tissues, circ-ANXA7 was elevated in DDP-resistant NSCLC tissues, clarifying that circ-ANXA7 was associated with DDP resistance in NSCLC (Fig. 1A).Subsequently, analysis of the clinicopathological characteristics was implemented.Likewise, in normal cells (HBE-1), NSCLC cell lines (A549 and H460) and DDP-resistant NSCLC cells (A549/DDP and H460/DDP), it was observed that circ-ANXA7 was the highest in DDP-resistant cells (Fig. 1B).
RNase R analysis and Act D analysis confirmed the annular characteristics of circ-ANXA7, showing that circ-ANXA7 was available to resist RNase R detachment, and its stability was better than linear ANXA7 mRNA (Fig. 1C-D).Additionally, circ-ANXA7 was primarily distributed in NSCLC cell cytoplasm (Fig. 1E).Kaplan-Meier analysis revealed that high circ-ANXA7 was associated with unpleasing overall survival in DDP-resistant NSCLC patients, as presented in Fig. 1F.
To sum up, circ-ANXA7 was stable and elevated in DDP-resistant NSCLC and might be implicated in NSCLC DDP resistance.
It was found that A549 cells had stronger DDP resistance (Fig. 2A) than H460 cells, and circANXA7 expression in A549 and A549/DDP cells was higher than that in H460 and H460/DDP cells (Fig. 1B), so A549/DDP cells were selected for subsequent studies.As presented in Fig. 2B, circ-ANXA7 was repressed in A549/DDP cells after transfection with sh-circ-ANXA7.Silence of circ-ANXA7 declined DDP IC 50 (Fig. 2C).Cell proliferation and apoptosis were subsequently examined, and it was found that circ-ANXA7 gene knockout inhibited the viability of A549/DDP cells (Fig. 2D-E), while the apoptosis rate increased significantly (Fig. 2F).In addition, Transwell analysis confirmed that circ-ANXA7 silencing effectively blocked A549/DDP cell migration and invasion (Fig. 2G-H).Western blot analysis of drug resistance-associated proteins (P-gp and GST-π) was performed, elucidating that P-gp and GST-π in A549/DDP cells were lowered after suppressing circ-ANXA7, as presented in Fig. 2I.
In general, repressing circ-ANXA7 was available to suppress NSCLC progression and strengthened DDP sensitivity.
All in all, loss of circ-ANXA7 interacted with DDP therapy, thereby restraining tumor growth in xenograft tumor models.

DISCUSSION
Globally, NSCLC is a deadly cancer with elevated morbidity and mortality (Cao et al., 2021).DDP is the most frequently-adopted chemotherapy drug for the treatment of NSCLC (Dong et al., 2019).Nevertheless, DDP resistance severely limits its clinical efficacy (Hong et al., 2020).Consequently, it was crucial to suppress DDP resistance for better treatment of NSCLC patients.It is reported that aberrant circRNA impacts NSCLC cancerization and chemotherapy resistance (Zhang et al., 2021).In this study, it was first discovered that repres-sion of circ-ANXA7 suppressed NSCLC progression and strengthened DDP sensitivity.Additionally, this study first verified the regulatory network of cir-ANXA7/miR-545-3p/CCND1.
Research has shown that the covalent closed structure of circRNAs makes them more stable in eukaryotes (Fu et al., 2021).Likewise, the data displayed that circ-  ANXA7 after RNase R and Actinomycin D treatment was more stable than linear ANXA7.circRNAs exert critical roles in multiple biological processes of NSCLC (Liu et al., 2021).For instance, in DDP-resistant NSCLC tissues and cells, circ_PIP5K1A expression is augmented, while silencing circ_PIP5K1A is available to restrain cancer progression and strengthen DDP sensitivity (Feng et al., 2021).Additionally, silencing circ_0076305 ameliorates the DDP sensitivity of NSCLC (Wang et al., 2021).In other words, circRNAs modulate cancer progression and DDP resistance.This research focused on a novel circrNA (Circ-ANXA7) derived from host gene ANXA7 chr10 (75138745-75138766).Expression analysis displayed that circ-ANXA7 was elevated in DDPresistant NSCLC tissues and cells, clarifying that circ-ANXA7 might participate in DDP resistance progression in NSCLC.As expected, it was discovered that circ-ANXA7 silencing inhibited the proliferative activity, migration, invasion, and DDP sensitivity of DDP resistant cells, but promoted apoptosis.Overall, the data have illustrated that circ-ANXA7 is a promoter of NSCLC progression and drug resistance.Nevertheless, circ-ANXA7 in patients' serum was not detected in this study.Notably, a recent report has clarified that exosomes-delivered hsa_circ_0014235 expedites DDP resistance and exacerbates NSCLC development via mediating miR-520a-5p/ CDK4 pathway (Xu et al., 2020).Consequently, it was a necessity to further examine circ-ANXA7 in NSCLC clinical serum samples in subsequent studies, which might offer novel data support for circ-ANXA7 as a promoter of NSCLC resistance.
As reported, the regulatory function of circRNA is associated with the miRNA/mRNA signaling network (Feng et al., 2021).Notably, it is reported that circRNAs participate in various human cancers via effectively targeting miRNA to mediate gene (Liu et al., 2022).For instance, silencing circ_0007385 restrains malignant behavior and DDP resistance of NSCLC cells via miR-519d-3p/HMGB1 axis (Ye et al., 2020).Circ_0076305 modulates STAT3 and DDP resistance of NSCLC cells via absorbing miR-296-5p as a sponge (Dong et al., 2019).Consequently, miR-545-3p was discovered to have a targeted binding site with circ-ANXA7.Foregoing studies have elucidated that miR-545-3p is silenced in NSCLC and performs as a tumor suppressor gene, restraining NSCLC progression and strengthening DDP sensitivity (Du et al., 2021;Li, Liu, and Qin 2020).Likewise, in this study, it was discovered that miR-545-3p was silenced in DDP-resistant NSCLC tissues and cells and performed as a tumor suppressor gene.Additionally, it was first discovered that miR-545-3p was competitively adsorbed by circ-ANXA7, and upregulating miR-545-3p reversed the inhibitory effect of circ-ANXA7 loss on NSCLC progression and DDP resistance.
miRNAs are available to repress specific proteins after transcription via combining with the 3'UTR of target mRNA (Pang et al., 2020).Consequently, the target genes of miR-545-3p were predicted, and CCND1 among numerous mRNAs has drawn our attention due to its relationship with DDP resistance (Zuo et al., 2021).CCND1 performs as a carcinogen in NSCLC, promoting proliferation, migration, invasion, and drug resistance of NSCLC cells, and may become a potential therapeutic target for NSCLC (Huang et al., 2020;Cui et al., 2020;Liu et al., 2020).Nevertheless, the mechanism of circ-ANXA7/miR-545-3p/CCND1 axis in NSCLC DDP resistance has not been explored.In this research, it was testified that elevated CCND1 was presented in DDPresistant NSCLC tissues and cells.Additionally, it was first discovered that miR-545-3p was implicated in DDP resistance in NSCLC via targeting CCND1.
In brief, circ-ANXA7 accelerated DDP resistance in NSCLC via the miR-545-3p/CCND1 axis, which might offer brand-new insights into the treatment of DDP resistance in NSCLC.Nevertheless, the potential involvement of downstream pathways was not considered in this study.In addition, future multicenter and animal studies are needed to further elucidate the role of circ-ANXA7 in DDP resistance in NSCLC.

Figure 1 .
Figure 1.Circ-ANXA7 is modulated in DDP-resistant NSCLC tissues and cells (A) RT-qPCR test of circ-ANXA7 in each tissue; (B) RT-qPCR examination of circ-ANXA7 in each cell; (C-D) RNase R and Actinomycin D determination of the stability of circ-ANXA7; (E) RT-qPCR test of the localization of circ-ANXA7 in A549/DDP cells; (F) Kaplan-Meier analysis of the survival prognosis of DDP-resistant NSCLC patients.Data were expressed as mean ± S.D. (Number of samples =3).*P<0.05.

Figure 2 .
Figure 2. Repression of circ-ANXA7 restrains NSCLC progression and strengthens DDP sensitivity (A) MTT analysis of IC 50 ; (B) RT-qPCR test of sh-circ-ANXA7 transfection efficiency; (C) MTT analysis of IC 50 of DDP in A549/DDP cells after transfection; (D-E) MTT and colony formation assay detection of cell proliferative activity; (F) Flow cytometry test of cell apoptosis; (G-H) Transwell assay test of cell migration and invasion; (I) Western blot detection of drug-resistance associated proteins (P-GP and GST-π).Data were expressed as mean ± S.D. (Number of samples=3).*P<0.05.

Figure 5 .
Figure 5. CCND1 is the functional target of miR-545-3p (A) Bioinformatics sites forecast of binding sites of CCND1 with miR-545-3p; (B-C) Luciferase activity assay, and RIP assay evaluation of the interaction of CCND1 with miR-545-3p; (D-G) RT-qPCR and Western blot test of CCND1 in tissues and cells; (H-I) Pearson correlation analysis assessment of the association of CCND1 with miR-545-3p and circ-ANXA7.(J) Western blot examination of CCND1 after transfection with sh-circ-ANXA7 or miR-545-3p mimic.Data were expressed as mean ± S.D. (Number of samples =3).*P<0.05..

Figure 6 .
Figure 6.Circ-ANXA7 impacts NSCLC progression and DDP resistance via miR-545-3p/CCND1 axis (A-B) RT-qPCR and Western blot examination of CCND1 in A549/DDP cells; (C) MTT analysis of IC 50 of DDP in A549/DDP cells; (D-E) MTT and colony formation assay detection of cell proliferative activity; (F) Flow cytometry test of cell apoptosis; (G-H) Transwell assay test of cell migration and invasion; (I) Western blot test of drug-resistance associated proteins (P-gp and GST-π).Data were expressed as mean ± S.D. (Number of samples =3).*P<0.05.
Table 2 elucidated no difference in age, tumor size, gender, and metastasis between DDP-resistant patients with non-resistant patients.Nevertheless, DDP resistant patients had high levels of circ-ANXA7, deep invasion, low rate of tumor metastasis, and poor differentiation.