Hsa_circ_0000199 facilitates chemo-tolerance of triple-negative breast cancer by interfering with miR-206/613-led PI3K/Akt/mTOR signaling

Increasing attentions have been paid to the role of circRNAs in the etiology of triple-negative breast cancer (TNBC), and we strived to figure out the association of circRNA AKT3/miRNA axis with TNBC chemo-resistance. Altogether 207 BC patients were divided into TNBC group (n=83) and non-TNBC group (n=124), and MCF-10A, MDA-MB-231, MDA-MB-468, SK-BR-3 and MCF-7 cell lines were prepared in advance. Expressions of AKT3-derived circRNAs and relevant miRNAs in the TNBC tissues and cell lines were determined by employing real-time polymerase chain reaction (PCR). It was indicated that hsa_circ_0000199 expression was higher in TNBC tissues than in non-TNBC tissues, and high hsa_circ_0000199 expression was predictive of large tumor size, advanced TNM grade, high Ki-67 level and poor 3-year survival of TNBC patients (all P<0.05). Furthermore, miR-613 and miR-206 were sponged and negatively regulated by hsa_circ_0000199 (P<0.001), and PI3K/Akt/mTOR signaling was depressed by si-hsa_circ_0000199 in TNBC cell lines (P<0.01). Ultimately, miR-206/miR-613 inhibitor reversed impacts of si-hsa_circ_0000199 on PI3K/Akt/mTOR signaling, proliferation, migration, invasion, chemo-sensitivity and autophagy of TNBC cells (all P<0.01). Conclusively, silencing of hsa_circ_0000199 enhanced TNBC chemo-sensitivity by promoting miR-206/miR-613 expression and deactivating PI3K/Akt/mTOR signaling, which was conducive to improving chemotherapeutic efficacy of TNBC patients.


INTRODUCTION
Triple-negative breast cancer (TNBC), making up 10%~20% of breast cancer (BC) cases [1], revealed clinical attributes of malignant invasion, aggressive lymph-node metastasis and high recurrence [2].Owing to shortages of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her-2), endocrino-and trastuzumab-based therapies that worked for other BC subtypes were no longer suitable for TNBC treatment [3].Instead, chemotherapies have been broadly applied to benefit TNBC patients, yet drug-tolerance rendered these strategies less efficacious than anticipated [4], which underscored the necessity of elucidating molecular mechanisms implicit in TNBC chemo-resistance.
Circular RNAs (circRNAs), originally mistaken as nonfunction products of RNA splicing, held potential to diagnose malignancies, and their dysfunction could powerfully drive progression of tumors [5], including BC [6], gastric cancer [7], glioma [8] and colorectal cancer [9].Specifically, high expression of circKIF4A was associated with elevated likelihood of TNBC onset [10], and survival of TNBC patients was prolonged when they carried low expression of circAGFG1 [11].Furthermore, knockout of circGFRA1 dampened proliferation and enabled apoptosis of TNBC cells [12], while adriamycin (ADM)-sensitivity of MCF7 cell line was encouraged after silencing of hsa_circ 0006528 [13].Despite the growing recognition that circRNAs mattered in BC etiology, few circRNA-centric signaling pathways were verified to account for intensified drugresistance in TNBC.
To bridge this gap, this investigation was intended to elucidate the contribution of circRNA AKT3-centric miRNA axes underlying TNBC etiology, which might help to address concerns over TNBC chemo-resistance.
Collectively, it was implied that miR-613 and miR-206 were sponged and down-regulated by hsa_circ_0000199 in TNBC.

MiR-206 and miR-613 disturbed influence of hsa_circ_0000199 on TNBC autophagy
Beclin1 and LC3-II were a couple of proteins indispensable to cell autophagy [31,32], and p62 was degraded in case of cell autophagy [33].Not only that, Atg5 expression was promoted during autophagy, and its combination with Atg12 could drive extension of autophageosome membrane [34].Here we observed that Beclin1, LC3-II and p62 expressions were evidently boosted, and p62 expression was down-regulated in MDA-MB-231 and MDA-MB-468 cell lines after transfection of si-has_circ_0000199 (P<0.001)(Figure 7A, 7B).Consistently, MDC-positive particles in the form of bright blue dots were abundantly present in AGING MDA-MB-231 and MDA-MB-468 cell lines of si-hsa_circ_0000199 group in comparison to si-NC group, and the number of MDC-positive particle was falling in si-hsa_circ_0000199+3-MA group as relative to si-hsa_circ_0000199 group (Supplementary Figure 2).si-hsa_circ_0000199 group (all P<0.01) (Figure 7A, 7B).Co-transfection of si-hsa_circ_0000199 and miR-206/miR-613 inhibitor engendered less MDA-positive particles than simply transfection of si-circ_0000199 (Figure 7C).

DISCUSSION
Despite with a 5-year survival of 90%, there were up to 41,760 American females dying of BC in 2019, covering 15% of all tumor deaths [35].TNBC, a BC subtype notorious for high odds of recurrence and metastasis [36], was managed principally by various chemotherapies, whose efficacy, however, was reduced owing to drug-resistance.Therefore, clarification of TNBC etiology was urgently required, and growing interests were sparked concerning the implication of circRNA-led miRNA network in TNBC.
Distinct from linear RNAs with the structure of 5'-cap and 3'-tail, circRNAs in the shape of closed rings were produced through back-splicing approach [37], which made it tough to degrad circRNA with exonuclease and thereby maintained circRNA stability.Thanks to this trait, circRNAs were capable of reflecting cancer progression sensitively, including bladder cancer [38], hepatocellular cancer [39], laryngeal cancer [40] and basal cell carcinoma [41].With regard to BC, hsa_circ_006054 combined with hsa_circ_100219 and has_circ_406697 excelled in diagnosing BC patients from healthy volunteers [42], and molecular results showed that proliferation of MDA-MB-231 cell line was boosted by circDENND4C in the oxygen-free context [43].Nonetheless, circRNAs available to differentiate BC subtypes (e.g.TNBC) were poorly known, let alone their sophisticated function in TNBC etiology [12,44].In this investigation, AKT3-derived hsa_circ_0000199 was found to specifically overexpress in TNBC (Figure 1), and its high expression was associated with clinical symptoms of TNBC patients, rather than the whole BC population (Tables 1,  2).However, whether this result could be generalized to other populations demanded more researches.
We speculated that different cell types used and distinct experimental procedures followed could account for this paradox, yet convincing evidence was entailed.Additionally, PI3K/Akt/mTOR signaling, downstream pathway of hsa_circ_0000199-miR-206/miR-613 axis in TNBC (Figure 4), was reported to stimulate tumor onset and to potentiate metastasis and proliferation of tumor (e.g.TNBC) cells [54].Moreover, phosphorylated mTOR was measurable in a larger share of TNBC patients than in non-TNBC patients, which stressed that PI3K/Akt/mTOR signaling could matter in TNBC as compared with other BC subtypes [55].Furthermore, rapamycin treatment, a common inhibitor of mTOR signaling, not merely strengthened anti-TNBC power of adriamycin in nude mice [56], but also improved paclitaxel's performance in fighting against TNBC [57], implying that it was practicable to raise TNBC chemosensitivity by attenuating PI3K/Akt/mTOR signaling.In addition, autophagy, which served bi-directional roles in tumors [58], was induced when PI3K/Akt/mTOR signaling was obstructed [28].This physiological change was likely to weaken tumor development by facilitating apoptosis of tumor cells, which also explained increased chemo-sensitivity of BC [59].Allowing for multiple roles performed by PI3K/Akt/mTOR signaling, it might be tenable that hsa_circ_0000199-miR-206/miR-613 axis controlled proliferation, migration, invasion, drug-resistance and autophagy of TNBC cells.

CONCLUSIONS
In conclusion, hsa_circ_0000199-miR-206/miR-613 axis pronouncedly disordered migration, invasion, chemoresistance and autophagy of TNBC cells by motivating PI3K/Akt/mTOR signaling (Figure 8), providing molecular foundations for developing TNBC treatments.However, several pitfalls should be addressed in later researches.Firstly, it was uncertain whether hsa_circ_ 0000199 was applicable in distinguishing TNBC from other BC subtypes among populations of other ethnicities.Secondly, although hsa_circ_0000199 expression was heightened in tumor tissues of TNBC-bearing mice models as compared with paired normal tissues (Supplementary Figure 3), we failed to uncover the effect of over/under-expressed hsa_circ_0000199 on tumor formation in TNBC mice models owing to technical obstacles.Last but not the least, miRNA networks that aided hsa_circ_0000199 to function oncogenetically in TNBC should be expanded, so as to deepen understanding of TNBC pathogenesis.Above all, all these challenges should be coped with in future.

Collection of BC samples
Two hundred and seven cases out of 210 primary BC patients (response rate: 98.57%) were recruited in Minhang Hospital affiliated to Fudan University, from December, 2012 to May, 2016.They were divided into TNBC group (n=83) and non-TNBC group (n=124) based on the immunohistochemical results, and they all underwent none of surgical puncture, immunity enhancement, chemotherapy and radiotherapy prior to surgery.The TNBC patients all exhibited negative expressions of ER, PR and Her-2, and the BC cases were graded according to TNM staging system revised by American Joint Committee on Cancer (AJCC)/Union International Center of Cander (UICC) (6 th edition) [60].The participants have signed informed consents, and this program was approved by Minhang Hospital affiliated to Fudan University and the ethics committee of Minhang Hospital affiliated to Fudan University.Additionally, BC tissues and adjacent normal tissues, after excision from patients during surgery, were split into pieces weighing around 0.1 g before storage at -80° C.

Methyl thiazolyl tetrazolium (MTT) assay to assess chemosensitivity of TNBC cells
MDA-MB-231 and MDA-MB-468 cell lines of logarithmic growth were seeded into 96-well plates at the concentration of 1×10

Cell counting kit-8 (CCK8) assay
Abiding by procedures detailed in the CCK8 kit (Sino-American Biotechnology, China), MDA-MB-231 and MDA-MB-468 cell lines were blended with 10 μl enhanced CCK8 reagent.After incubation at 37° C for 1 h, optical density (OD) of TNBC cells was evaluated on microplate reader (model: iMark, BioRad, USA) at the wavelength of 450 nm.

Cell migration
The upper transwell chamber (Corning, USA) was inoculated by 1×10 ´ AGING were eliminated with a cotton rub, and pictures of 5 views were taken to count average cell number with inverted microscope (Nikon, Japan).

Cell invasion
After dilution by serum-free and high-glucose DMEM (Biological Industries, USA) at the ratio of 1:8, 100 μl Matrigel (Corning, USA) was paved onto the center of upper transwell chamber (Corning, USA).Then 1×10 5 TNBC cells were supplemented onto the coagulated Matrigel, and 700 μl 10% FBS-inclusive high-glucose DMEM was poured into the lower chamber (Corning, USA).Twenty-four ours later, MDA-MB-231 and MDA-MB-468 cell lines were managed by 10% methanol for 20 min, followed by dyeing with 0.5% crystal violet for 30 min.Eventually, TNBC cells that hardly penetrated the upper chamber were wiped off, and 5 fields were randomly selected to count cell number under inverted microscope (Nikon, Japan).

Monodansylcadaverine (MDC) staining to determine autophagic condition of TNBC cells
After digestion by pancreatin to a density of 3×10 4 /ml, MDA-MB-231 and MDA-MB-468 cell lines at the logarithmic growth phase were inoculated into 12-well plates until 70% confluence.Each cell sample was evenly mixed with 10 μl MDC solution (Sigma, USA), which was then left in the darkness for 40 min.After centrifugation at 1000 g for 5 min, the TNBC cells were re-suspended in 100 μl PBS, and they were photographed under fluorescent microscope (Olympus, Japan).

Real-time quantitative polymerase chain reaction (PCR)
Total RNAs were extracted from BC tissues and cell lines by addition of TRIzol reagent (Invitrogen, USA).Integrity of the RNAs was confirmed through agarose gel electrophoresis (AGE), and their concentration and purity were determined with spectrophotometer (model: SmartSpec Plus, Bio-Rad, USA).Subsequently, the RNAs were reversely transcribed into cDNAs (TransGen Biotech, China), and cDNAs were amplified (Applied Biosystems, USA) on the real-time PCR instrument (model: 9300, Bio-Rad, USA) following procedures of: 1) pre-denaturation at 95° C for 3 min, and 2) 40 cycles of denaturation at 95° C for 5 s, annealing at 60° C for 30 s and extension at 72° C for 30 s. Primers of circRNAs and miRNAs were arranged in Supplementary Tables 1, 2, and their relative expression was calculated through 2 -ΔΔCt approach [61].Expressions of circRNAs were normalized to that of GAPDH, and U6 was set as the internal reference of miRNAs.

Statistical analyses
All data were analyzed with SPSS 17.0 software (IBM Corporation, USA).Differences among categorical variables (n) were discerned using chi-square test, and continuous variables [mean ± standard deviation (SD)] were compared through student's t test or single-factor analysis of variance (ANOVA).Kaplan-Meier survival curves were plotted, and log-rank test was applied to identify statistical difference between groups.Coxproportional hazard model was also established to screen out variables that were predictive of TNBC patients' survival.It was statistically significant when P value was less than 0.05.AGING