Downregulation of circ-PSMB6 suppresses NSCLC progression and metastasis through sponging miR-532-5p and regulating EZH1 expression


 Background: Accumulating reports showed how circular RNAs (circRNAs) act importantly during tumor progression via regulating gene expression, but regulatory mechanisms remain largely unknown. Current investigation clarified circRNA regulatory mechanisms in non-small cell lung cancer (NSCLC).Methods: High-throughput sequencing and quantitative reverse transcription polymerase chain reaction (RT-qPCR) detection were utilized to explore circRNA expression in NSCLC tissues and cells. Our lab did statistical analyses and luciferase reporter analysis to validate correlations between circRNA, miRNA and gene expression. We transfected NSCLC cells with different vectors, and transwell migration, Cell Counting Kit-8 (CCK-8) proliferation along with colony formation assays were performed. In vivo tumorigenesis and metastasis assays were utilized to validate the circRNA role in NSCLC.Results: Data illustrated that hsa_circ_0041595 (circ-PSMB6) incremented in NSCLC cell lines and tissues, while circ-PSMB6 downregulation suppressed NSCLC cell proliferation and invasion in vitro and in vivo. Bioinformatics analysis and luciferase reporter data verified that miR-532-5p and Enhancer Of Zeste 1 Polycomb Repressive Complex 2 Subunit (EZH1) were circ-PSMB6 downstream targets in NSCLC cells. Overexpression of EZH1 or miR-532-5p inhibition reversed NSCLC cell invasion and proliferation after silencing circ-PSMB6. Further experiments discovered that circ-PSMB6 can influence cancer stem cell differentiation by regulating miR-532-5p/EZH1.Conclusions: Taken together, we found that circ-PSMB6 suppressed NSCLC metastasis and progression via sponging miR-532-5p and regulating EZH1 expression.

Results: Data illustrated that hsa_circ_0041595 (circ-PSMB6) incremented in NSCLC cell lines and tissues, while circ-PSMB6 downregulation suppressed NSCLC cell proliferation and invasion in vitro and in vivo. Bioinformatics analysis and luciferase reporter data veri ed that miR-532-5p and Enhancer Of Zeste 1 Polycomb Repressive Complex 2 Subunit (EZH1) were circ-PSMB6 downstream targets in NSCLC cells. Overexpression of EZH1 or miR-532-5p inhibition reversed NSCLC cell invasion and proliferation after silencing circ-PSMB6. Further experiments discovered that circ-PSMB6 can in uence cancer stem cell differentiation by regulating miR-532-5p/EZH1.

Background
Lung cancer is identi ed by high morbidity and poor prognosis. NSCLC is the main class of lung cancer, which accounts for about 85% lung cancer cases [1]. We now know that smoking is the predominant reason for NSCLC [2]. Since NSCLC shows no early apparent clinical symptoms and screening applications are not e cient, majority patients diagnosed with NSCLC are in an advanced stage with poor prognosis [3]. Many clinical investigations have shown that metastasis hinders NSCLC cancer treatment.
Therefore, a better understanding the mechanisms underlying metastasis is crucial for treating NSCLC effectively.
Circular RNAs (circRNAs) are a class of endogenous and conserved non-coding RNA (ncRNA) that form covalently closed continuous loops via back-splicing with no 3' or 5' end [4]. More and more studies discovered a substantial effect of ncRNAs including circRNAs, long ncRNAs and microRNAs (miRNAs) on tumor development [5,6]. For example, circFGFR1 increments progression and anti-PD-1 resistance through sponging miR-381-3p in NSCLC cells [7]. The circ-ABCB10 promotes NSCLC proliferation and inhibits cell apoptosis by repressing KISS1 [8]. The circGFRA1 promotes NSCLC progression through miR-188-3p sponging [9]. Whereas circRNA roles in NSCLC progression are still unclear. Our study was thus aimed at verifying the potential effects and underlying mechanisms of circRNAs in NSCLC progression.  Total RNA isolation and RT-qPCR Our lab employed TRIzol reagent to obtain total RNA from tumor cells and tissues following manufacturer's protocol. Our lab measured RNA sample concentration and purity at absorbances of 230, 260, and 280 nm with NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scienti c, Wilmington, USA). We regarded OD 260 / 280 and OD 260 / 230 ratios in 1.8~2.1 and >1.8 as acceptable.

Transwell migration assay
Our lab assessed cell migration through Costar ® Transwell ® cell culture inserts (Corning Inc., Corning, NY, USA) following instructions. After incubating the cells for 1 d, our lab erased cells on Transwell chamber upper surfaces via cotton swabs. Technician xed cells on lower surfaces in methanol for 10 minutes and stained them with Crystal Violet. Our team counted and photographed cells in 5 elds that randomly selected.
Plate colony formation assay PC9 and A549 cells after different treatments were resuspended and counted, and we seeded 200 cells/well in plates with six wells. Our lab incubated cells for 2 weeks, with the medium changed every 3 days. We took photographs with a uorescence microscope before ending the experiment, then washed the cells twice with PBS. The cells were stained in 500 µL Giemsa dye for 10-20 min, washed three times with ddH 2 O and photographed with a digital camera.

CCK-8 assay
We determined proliferation rates 1, 2, and 3 days after transfection using Cell Counting Kit-8 (CCK-8) assays following instructions. Technician incubated cells in 10% CCK-8 solution diluted in normal culture medium at 37°C until visual color conversion occurred. We read every well absorbance using microplate reader at 570 nm.

Dual-luciferase reporter assay
Putative miR-532-5p binding sites in target gene EZH1 and in circ-PSMB6 were cloned into the psi-CHECK vector (Promega, Madison, Wisconsin, USA). Wild type (Wt) or mutated (Mut) 3'-UTR sequences from EZH1 or Wt and Mut miR-532-5p binding sites from circ-PSMB6 were inserted downstream from re y luciferase as the primary luciferase signal and were designated EZH1-Wt/circ-PSMB6-Wt and EZH1-Mut/circ-PSMB6-Mut. psi-CHECK vector itself supplied a Renilla luciferase signal as a normalization factor to compensate for the differences between transfection and harvested e ciencies. Our team then transfected 293T cells with these constructs using Lipofectamine 2000 and detected Renilla and re y luciferase activities 1 d after transfection with Dual-Luciferase Reporter Assay System (Promega) utilizing luminometer (Molecular Devices, U.S.A.). We processed relative Renilla luciferase activity following the manufacturer instructions.
Tumor sphere formation assays PC9 and A549 cells were harvested and resuspended as single cells in serum-free medium. Following cell counting, our lab seeded 200 cells/well in 200 μL serum-free medium in plates with 96 wells, 10 wells/group, altering medium every 2 d. Our lab obtained ve images from randomly selected regions in each well group using camera-equipped microplate reader (Leica, Wetzlar, Germany) and computed sphere percent as number of spheres/200.

Cell cycle detection
Our team dissociated cells in logarithmic growth phase with 0.25% trypsin, resuspended them in PBS and xed them in ice-cold 70% ethyl alcohol overnight at 4°C. Our team centrifuged cells at 1000 rpm for ve minutes, resuspended them in 50 μL RNase A and incubated them at 37°C for half of an hour. We added 400 μL propidium iodide (PI) to suspension for 0.5 h, followed by ow cytometry (BD Bioscience, CA, USA).

Animal studies
To detect the circ-PSMB6 role in lung cancer metastasis model, we intravenously injected 1×10 6 stable lentiviral-mediated circ-PSMB6-silenced PC9 cells (sh-circ-PSMB6) or negative control (NC) PC9 cells into male nude mice (Chinese Science Academy, Shanghai, China) via tail vein. After one month, our team analyzed PC9 cell metastasis through bioluminescence imaging following intravenous luciferin injection (150 mg luciferin/kg body weight) into tails.
For xenograft assays, our team subcutaneously injected 1×10 6 modi ed or control PC9 cells (sh-circ-PSMB6 or Wt,) into the male nude mice right ank. Tumor volumes were calculated as 0.5 × length × width 2 at time points indicated, and technician excised all tumors 4 w after injection. All animals were treated according to instructions from Animal Care Committee in Shanghai University of Medicine and Health Sciences.

Immunohistochemical analysis
Our team xed tumor tissue samples in 10% formalin solution, embedded them in para n and sliced them into 5-μm-thick sections, which we stained with antibodies against Ki67 to validate cell proliferation, using AxioPhot light microscope (Carl Zeiss AG, Oberkochen, Germany) equipped with digital camera Statistical analyses Statistician compared differences between any 2 groups by unpaired or paired 2-tailed t-test. Bioinformatician leveraged Pearson's correlation coe cient to characterize correlations between groups. Bioinformatician denoted data as means ± standard deviation. Our team considered probability (p) values < 0.05 statistically signi cant. We employed GraphPad Prism (GraphPad Software, La Jolla, CA, USA) for statistical analyses.

Results
Differential expression of circRNAs in NSCLC.
High-throughput sequencing found that 1308 circRNAs were upregulated and 1530 circRNAs were downregulation in NSCLC tissues (T) comparing to adjacent non-tumorous tissues (T), shown as volcano plots in Fig. 1A. The length distribution of circRNAs were mostly concentrated below 1,000 bp (Fig. 1B).
circ-PSMB6 expression increased in NSCLC cells and circ-PSMB6 knockdown caused cell cycle arrest in G2 phase.
Bioinformatics analyses indicated that hsa_circ_0041595 was located in chr17:4699456-4701790 and originated from an exon in the gene encoding Proteasome 20S Subunit Beta (PSMB6) containing 826 bp. Therefore, hsa_circ_0041595 was named circ-PSMB6 ( Fig. 2A). RT-qPCR assays showed that circ-PSMB6 expression in NSCLC cells lines PC9, H1299, A549, H1650 and H1975 was increased comparing to BEAS-2B control cells. Because PC9 and A549 cells had the highest circ-PSMB6 expression levels, which were chosen for the study (Fig. 2B). We constructed siRNA against circ-PSMB6 and found that circ-PSMB6 expression was signi cantly decreased when circ-PSMB6 was silenced in both PC9 and A549 cells (Fig. 2C). Flow cytometric analysis showed that circ-PSMB6 silencing caused cell cycle arrest in the G2 phase (Fig. 2D), suggesting that circ-PSMB6 plays a role in NSCLC progression.
Downregulating circ-PSMB6 inhibited cell metastasis and proliferation in vivo and in vitro. CCK8 (Fig. 3A-B) and clonal formation assays ( Fig. 3C and 3D) demonstrated that circ-PSMB6 downregulation inhibited cell proliferation in PC9 and A549 cells. In nude mouse xenografts, circ-PSMB6 silencing suppressed PC9 tumor growth in both volume and weight (Fig. 3E-G). Immunohistochemistry also illustrated that circ-PSMB6 downregulation inhibited Ki67 expression. These studies thus demonstrated that circ-PSMB6 downregulation inhibited tumor growth and cell proliferation both in vivo and in vitro.
Transwell assays were used to examine PC9 and A549 cell migratory capacity. Results indicated that circ-PSMB6 silencing suppressed cell migration ( Fig. 4A and 4B). Live imaging showed the circ-PSMB6 effect on NSCLC metastasis regarding PC9 cells 4 weeks after tail intravenous injection. circ-PSMB6 silencing inhibited the number of metastatic foci in lung tissues based on hematoxylin/eosin staining (Fig. 4C-E).
Bioinformatics analysis showed that circ-PSMB6 could interact with a series of miRNAs. A comprehensive Venn diagram analysis utilizing the Starbase, circBank and circInteractome databases found that only miR-532-5p and miR-1179 could interact with circ-PSMB6 in a common region that was identi ed by all three databases (Fig. 5A). RT-qPCR analyses demonstrated that miR-532-5p expression decremented signi cantly in NSCLC tissues (Fig. 5B). We then constructed a luciferase reporter vector containing Wt or Mut circ-PSMB6 sequence and co-transfected it with miRNA mimics into 293T cells. Data illustrated that only miR-532-5p could decrease uorescein intensity signi cantly, advising that miR-532-5p was the circ-PSMB6 downstream target (Fig. 5C). Luciferase reporter data further con rmed that miR-532-5p inhibited luciferase activity in Wt constructs, yet not in Mut cell lines (Fig. 5D), demonstrating that miR-532-5p was the circ-PSMB6 target.
Overexpression of EZH1 or miR-532-5p inhibition reversed NSCLC cell migration and proliferation after silencing circ-PSMB6 via differential regulation of cancer stem cells.
CCK8 and clonal formation assays showed that overexpression of EZH1 or inhibition of miR-532-5p reversed proliferation of both PC9 and A549 cells after circ-PSMB6 was silenced (Fig. 6A-E). Transwell assays to assess cell migration also showed that overexpressing of EZH1 or inhibiting miR-532-5p blocked the migratory capacity of both PC9 and A549 cells after silencing circ-PSMB6 (Fig. 6F-H).
Western blots indicated that overexpression of EZH1 or inhibition of miR-532-5p slowed tumor sphere formation by both PC9 and A549 cells after circ-PSMB6 was silenced (Fig. 7), suggesting that these effects were mediated by differential regulation of cancer stem cells.

Discussion
The circRNAs belong to a class of recently discovered ncRNAs, and a number of reports show that they may be applied as diagnostic biomarkers as well as therapeutic targets in various cancers. circ_0078767 suppresses NSCLC through protecting RASSF1A expression through sponging of miR-330-3p [10]. circ-NDUFB2 inhibits NSCLC progression by destabilizing IGF2BPs and activating anti-tumor immunity [11]. The present investigation discovered that circ-PSMB6 expression incremented in NSCLC tissues comparing with non-tumorous tissues. circ-PSMB6 downregulation arrested cell cycles of A549 and PC9 cells in G2 phase, suppressed cell proliferation and slowed invasion in in vitro and in vivo experiments. These data demonstrated that circ-PSMB6 may facilitate the progression of NSCLC.
More and more studies suggested that circRNAs can act as miRNA sponges to regulate gene expression [12,13]. In this study, we found that circ-PSMB6 interacted with miR-532-5p, which was con rmed by luciferase report analysis. Previous research has revealed that miR-532-5p enhances breast cancer migration and proliferation via targeting RERG [14] and suppresses lung cancer cell invasion and migration via inhibiting CCR4 [15]. miR-532-5p is also prognostic marker which suppresses cell invasion and proliferation through targeting TWIST1 in epithelial ovarian cancer [16]. The present study found that downregulation of circ-PSMB6 promoted miR-532-5p expression, while miR-532-5p inhibition restored invasive and proliferative capacity of NSCLC cells when circ-PSMB6 was silenced.
Further results indicated that miR-532-5p could act with EZH1 3'UTR and suppress EZH1 expression at mRNA level. Luciferase reporter experiments demonstrated that EZH1 was miR-532-5p target. Downregulating miR-532-5p restored EZH1 expression after circ-PSMB6 was silenced. Overexpressing EZH1 reversed the decrease in proliferation and invasion after downregulation of circ-PSMB6. The zeste (EZH) 2 enhancer functions importantly in NSCLC progression. EZH2 functions critically in tumorigenesis and cancer progression via epigenetic gene silencing and chromatin remodeling [17,18]. More reports validated that EZH2 overexpression occurs in various human malignancies such like breast, bladder, esophageal, oral, hepatocellular, gastric, colon, prostate, and endometrial cancers [19][20][21]. Elevated EZH2 expression has been correlated with advanced stages of ovarian cancer [22]. Our current investigation suggested that EZH1 overexpression or miR-532-5p inhibition reversed NSCLC cell proliferation and migration after circ-PSMB6 was silenced via the differential regulation of cancer stem cells.

Conclusion
In summary, our study demonstrated that downregulation of circ-PSMB6 expression decreased NSCLC cell proliferation and invasions via miR-532-5p/EZH2 signaling regulation. The results inferred that circ-PSMB6 is potential diagnostic biomarker of NSCLC, and suggested that drugs targeting circ-PSMB6 may play a promising role in NSCLC treatment.

Declarations
Ethics approval and consent to participate None.

Consent for publication
All authors have agreed to publish this manuscript.

Availability of data and material
The data generated or analyzed during this study are included in this article, or if absent are available from the corresponding author upon reasonable request.

Competing interests
The authors declare that they have no competing interests.     Overexpression of EZH1 or inhibit miR-532-5p reversed NSCLC cells proliferaion and migration after silence circ-PSMB6. (A and B) CCK8 detection show the proliferation ability of PC9 and A549 cells. The data are expressed as the mean ± SD. ***p < 0.001 vs NC. (C-E) Clone formation assays show the proliferation ability of PC9 and A549 cells. The data are expressed as the mean ± SD. ***p < 0.001 vs NC. ###P<0.001 vs. si-circ-PSMB6. (F-H) Transwell detection show the invasion and migration of PC9 and A549 cells. The data are expressed as the mean ± SD. ***p < 0.001 vs NC. ###P<0.001 vs. si-circ-PSMB6.