CircKEAP1 serves as a ceRNA to suppress lung adenocarcinoma progression by activating KEAP1 signal pathway

Background Circular RNAs (circRNAs) are widely expressed noncoding RNAs, and plays a key role in the biological function of competitive endogenous RNA (ceRNA) network in various human diseases, especially in cancer. However, the regulatory roles of circRNAs in lung adenocarcinoma (LUAD) remains largely unknown. The expression proles of circRNAs in LUAD tissues and adjacent non-tumor tissues were analyzed by Agilent Arraystar Human CircRNA microarray. The level and prognostic values of circKEAP1 in tissues and cancer cell lines were determined by quantitative real-time PCR. Then, the effects of circKEAP1 on tumor growth were investigated by functional experiments in vitro and in vivo. Mechanistically, dual luciferase reporter assay, RNA pull-down and RNA immunoprecipitation experiments were performed to conrm the interaction between circKEAP1 and miR-141-3p in LUAD. both in vitro and repression for its target gene KEAP1, which activated the KEAP1/NRF2 signal pathway, and nally suppress the cell proliferation. ndings suggest inhibits progression through circKEAP1/miR-141-3p/KEAP1 axis and


Introduction
LUAD is the most common histopathological subtype of lung cancer, which is the most commonly diagnosed cancer and the leading cause of cancer death worldwide 1 . The morbidity of LUAD is continuously raised during the past years, especially in women, never-smokers and young adults 1 . The overall 5-year survival of LUAD is less than 20% due to the lack of effective early diagnosis and treatment options 2 . Therefore, a more comprehensive understanding of the molecular mechanisms of LUAD is important to develop reliable early biomarkers and novel therapies for LUAD. miRNA was a novel kind of about 22nt non-coding RNAs which could repress gene expression by binding with their target mRNAs 3 . It is well known that the downregulation of some tumor-suppressive genes in cancer may cause by the increase of miRNAs (named as oncomiR), whereas the decrease of tumorsuppressive miRNAs (named as tumor suppressor miRNAs) lead to the upregulation of oncogenes 4 .
CircRNAs is a novel class of endogenous RNAs, which have been discovered ubiquitously expressed in mammalian cells and played an important role in the regulation of gene expression 5 . CircRNAs are characterized by the covalently closed loop structure, which result to resistant to the degradation of exonuclease RNase R 5 , and used to be just considered as a by-product of a splicing error for a long time.
However, numerous of reports recently revealed circRNAs could act as miRNA sponges, protein sponges, transporters or scaffolds, which were in turn involved in the initiation and progress of a variety of diseases, especially cancers 5,6 . Consequently, circRNAs that function as sponge for miRNA are likely to be involved in cancer 7 . In LUAD, several circRNAs have been revealed to be signi cantly dysregulated 8,9,10,11,12,13,14 and could act as ceRNA to sponge oncomiR or tumor suppressor miRNAs 8,9,13,15,16,17 . For instance, circHIPK3 was found to modulate autophagy by sponging miR-124-3p 15 . Circ100146 could directly bind to miR-361-3p and miR-615-5p to promote the progress of non-small cell lung cancer 13 .
CircPTK2 functions as a sponge of miR-429/miR-200b-3p to inhibit TGF-β-induced EMT and LUAD cell invasion by relieving the repression of miR-429/miR-200b-3p for TIF1γ 9 . CircPRKCI was con rmed to act as a sponge for both miR-545 and miR-589 and abrogated their suppression of the protumorigenic transcription factor E2F7 to promote tumor growth in LUAD 17 .
In this study, the expression pro le of circRNAs in LUAD was investigated by Human CircRNA microarray V1 and a novel LUAD-related circRNA circKEAP1 was identi ed to be signi cantly downregulated in LUAD tissues. Subsequently, the function and molecular mechanism of circKEAP1 in LUAD development and progression was explored. As the results in the Human CircRNA microarray, the data showed circKEAP1 was remarkably downregulated in LUAD tissues, compared to the adjacent non-cancerous tissues. Additionally, circKEAP1 could inhibit cell proliferation by acting as a sponge for miR-141-3p to relieve miR-141-3p repression for its target gene KEAP1, which in turn downregulate the expression of NRF2 and repress the NRF2 signal pathway. In summary, our data show that circKEAP1 might act as a tumor suppressor via miR-141-3p-KEAP1-NRF2 signaling in LUAD.

Method And Materials
Patients' characteristics CircRNA and miRNA expression pro les for seven paired LUAD cancer tissues and adjacent normal tissues were generated using the Agilent-069978 Arraystar Human CircRNA microarray and NanoString nCounter Human miRNA Expression Assay. The clinicopathological features of these seven patients are described in Table s1. We collected 105 cancer tissues and paired distal normal tissues from patients who were rstly diagnosed with LUAD without any treatment at the Harbin Medical University Cancer Hospital (Harbin, China). The clinicopathological features are described in Table s2. The Ethics Committee of the Harbin Medical University Cancer Hospital authorized the study, and we conducted it in conformity to the Declaration of Helsinki.
RNA/ gDNA extraction and RT-PCR/ qRT-PCR assay Total RNAs were isolated by TRIzol reagent (Takara, Dalian, China) according to the manufacturer's instruction. gDNA was isolated by Genomic DNA Isolation Kit (Sangon Biotech, Shanghai, China). The quality and quantity of RNA and DNA were detected by Nanodrop 2000 spectrophotometer (Thermo Fisher Scienti c, USA). The nuclear and cytoplasmic fractions were puri ed by PARIS Kit (Ambion, Life Technologies). RNA was reverse transcribed by HiScript II Q RT SuperMixfor qPCR (+gDNA wiper) (Vazyme, Nanjing, China). The AmpliTaq DNA Polymerase (Life Technologies) was used for PCR. The 2% agarose gel electrophoresis was performed to observe the cDNA and gDNA PCR products. AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, China) was used for qRT-PCR, and GAPDH was used to normalize the level of circRNA and mRNA. Hydrolysis probe-based RT-qPCR assay of miRNA was performed according to the manufacturer's instructions (Applied Biosystems). The miRNA level was normalized by small nuclear U6. Primers are listed in Table s3.

RNase R treatment
Total RNA (about 2mg) was incubated with 5 U/μg RNase R (Epicentre Technologies) for 30 min at 37 °C. Then, the RNA was puri ed by RNeasy MinElute Cleaning Kit (Qiagen) according to the the manufacturer's protocols.
Actinomycin D assay BEAS-2B cells were exposed to 2 μg/ml actinomycin D (Sigma) at indicated time point. Subsequently, the cells were harvested to extract the total RNA, and the stability of circKEAP1 and KEAP1 mRNA was analyzed using qRT-PCR.

Vector construction and cell transfection
To overexpress circKEAP1, the full-length cDNA of circR-KEAP1 was synthesized and then cloned into pLCDH-ciR plasmid (Geneseed, Guangzhou, China). For luciferase reporter vector, the sequence of circKEAP1, mutated circKEAP1 (the seed sequence mutated from UGGUGUU to ACCACAA for binding site 1, and CAGCGUUG to GUCGCAAC for binding site 2), KEAP1 3'UTR and mutated KEAP1 3'UTR (the seed sequence mutated from CAGUGUU to GUCACAA ) was synthesized and then cloned into the pGL3promoter vector (Geneseed, Guangzhou, China). The Dual Luciferase Assay Kit (Promega, Madison, WI, USA) was used to examine the luciferase activity accordance to the manufacturer's protocols. SiRNAs of circKEAP1, miRNA mimics, miRNA inhibitors and corresponding negative control (NC) were synthesized by GenePharma (Shanghai, China). Cells were transfected using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instruction.

Cell proliferation assays
The proliferation activity of A549 cells was tested by both Cell-Light™ EdU DNA Cell Proliferation Kit

Western blot analysis
The protein extraction reagent (Thermo Scienti c) with a cocktail of proteinase inhibitors (Roche Applied Science, Switzerland) was used to isolate the total protein from cells or tissue samples. Equal amount of total protein was separated by 10% SDS-PAGE and transferred onto a PVDF membrane. Then, the membranes were blocked with 5% skimmed milk powder and incubated the membranes with primary antibodies at 4 °C overnight and then incubated with secondary antibodies at room temperature for 2 h.

Animal experiments
Stably over-expressed cell lines were established by transfecting A549 cells with circKEAP1 overexpressed plasmid or control plasmid and selected with puromycin. Then, we suspended 10 6 A549 cells in 100 μl PBS and injected into the right ank of 6-week-old male BALB/c nude mice. We measured the volume of tumors every ve days. After 20 days later, the mice were sacri ced and the tumors were collected for further analysis.

Statistical analyses
Adequate sample size was determined according to the previous studies that performed analogous experiments. A two-sided test was applied. The raw data applying t-test was normally distributed. Data are expressed throughout the manuscript as mean±SD. The SPSS 18.0 software was performed to the statistical analyses, and the GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA) was used to generate the graphs. A P-value <.0.05 was regarded as statistically signi cant.

CircKEAP1 is downregulated in LUAD
The expression pro le of circRNAs in seven paired samples of cancer tissues and adjacent normal tissues from LUAD patients was analyzed by Human CircRNA microarray. There were 32 dysregulated circRNAs with p-value < 0.05, of which 15 circRNAs were downregulated and 17 circRNAs were upregulated ( Fig. 1A and B and Table s4). Since the downregulated circRNAs in cancer tissues might play tumor suppressive roles and could serve a novel method to treat cancer, the 15 downregulated circRNAs were chosen for furth study. Firstly, we analyzed these downregulated circRNAs in another two circRNA expression pro le (GSE112214 and GSE101586) of LUAD in GEO database. The results showed hsa_circRNA_104126 and hsa_circRNA_102442 were signi cantly downregulated in all the three circRNA expression pro le (Fig. 1C). Then, the two circRNAs were determined in the seven paired samples of cancer tissues and adjacent normal tissues from LUAD patients by qRT-PCR. Consistent with the results of circRNA expression pro le, hsa_circRNA_104126 and hsa_circRNA_102442 were downregulated in the cancer tissues (Fig. 1D). Compared to the hsa_circRNA_104126, the expression level of hsa_circRNA_102442 in the normal tissues was much higher (Fig. 1D).
Hsa_circRNA_102442 (termed circKEAP1) is located at chr19: 10610070-10610756, and derived from exon2 of KEAP1 gene with a length of 686nt by alternative splicing (Fig. 2A). We ampli ed the backspliced junction of circKEAP1by divergent primers and con rmed by Sanger sequencing (Fig. 2A). Then, we performed Northern blot analysis by a probe targeted the back-spliced junction, and the result showed that circKEAP1 could be observed at 686nt (Fig. 2B). Additionally, PCR analysis for reverse-transcribed RNA (cDNA) and genomic DNA (gDNA) showed that divergent primers could amplify products from cDNA of adjacent normal tissues but not from cDNA of cancer tissues and gDNA (Fig. 2C). Next, the expression level of circKEAP1 in another 105 paired LUAD samples was analyzed by quantitative reverse transcription PCR (qRT-PCR). As showed in Fig.2D, the expression of circKEAP1 was obviously decreased in cancer tissues (Fig. 2D).
The same results were revealed in cell experiments. CircKEAP1 was ampli ed from cDNA of the human normal lung epithelial cell line BEAS-2B, but not from cDNA of LUAD cancer cell line A549 and gDNA (Fig.  2E). Subsequently, we treated the BEAS-2B cells with Actinomycin D (an inhibitor of transcription) to explore the stability of circKEAP1 and KEAP1. As showed in Figure 2F, the half-life of circKEAP1 transcript exceeded 24 h and was much longer than KEAP1. Finally, qRT-PCR analysis was conducted for nuclear and cytoplasmic circKEAP1 to observe cellular localization of circKEAP1. As showed in Fig.1H, circKEAP1 transcript was preferentially located in the cytoplasm (Fig. 2G).
Taken together, circKEAP1 mainly existed in the cytoplasm, and signi cantly decreased in LUAD cancer tissues.

circKEAP1 inhibits tumor growth in vivo and in vitro
To study the function of circKEAP1 in LUAD progression, we synthesized the full-length cDNA of circKEAP1 and cloned into the pLCDH-ciR plasmid, which contained a front and back circular frame. We found that circKEAP1 plasmid could successfully increase circKEAP1 expression in A549 cells (Fig. 3A).
A nude mice xenograft model by implanting A549 cells transfected with control plasmid or circKEAP1 plasmid was established to identify the effect of circKEAP1 on tumor growth in vivo. The A549 cells were subcutaneously injected into the right ank of 6-week-old male BALB/c nude mice. The tumor volumes were monitored from the 5 days after A549 cell injection, and the mice were sacri ced after 20 days. We found overexpression of circKEAP1 drastically suppress tumor growth of A549 cells (Fig. 3B). The tumor weights were remarkably decreased by circKEAP1 (Fig. 3C and D). As showed in gure 3E, both H&E staining and Ki-67 staining of these tumors showed decreased cell mitosis and lower percentage of proliferative cells in the circKEAP1 overexpression group. As the previous results in the cells (Fig. 3A), the expression level of circKEAP1 was much higher in the circKEAP1 overexpression group, compared to the control group (Fig. 3F). These data indicated that circKEAP1 repressed tumor growth in vivo. To identify the effect of circKEAP1 on cancer cell proliferation in vitro, the EDU assay and CCK-8 assay were performed to the A549 cells transfected with control plasmid or circKEAP1 plasmid. As the results showed in Fig. 3G and H, both the EDU assay and CCK-8 assay revealed circKEAP1 signi catly suppressed the cancer cell growth in vivo.
In summary, these ndings suggest that circKEAP1 could repress the tumor growth in vivo and in vivo.

circKEAP1 act as a sponge for miR-141-3p
Recently, circRNA has been increasingly found to be involved in cancer by releasing downstream molecules by competing with miRNA 5, 6 . As showed in Fig.2G, circKEAP1 was mainly located in the cytoplasm (Fig. 2G). CircKEAP1 was speculated to be severed as a miRNA sponge. In order to verify the hypothesis, we rstly analyzed the miRNA expression pro les of the seven paired LUAD cancer tissues and non-tumor tissues by Human miRNA Expression Assay. The volcano plot showed signi cantly different pro les of miRNAs between the seven paired samples of LUAD and adjacent normal tissues ( Fig. 4A and Table s6). Then, we predicted the potential binding sites of miRNAs in the circKEAP1by Targetscan 18 , and found a total of 135 miRNAs were identi ed as potential targets of circKEAP1 (Table  s7). As the circKEAP1 has been found to be downregulated in LUAD cancer tissues, compared to adjacent normal tissues (Fig. 2D), the miRNA which was upregulated in the LUAD tissues and had the binding site with circKEAP1 was choose for further study. The resulted showed 13 miRNAs, including miR-141-3p, miR-106b-3p, miR-93-3p, miR-21-3p, miR-19b-3p, miR-29b-5p, miR-3445-5p, miR-375 and miR-200c-3p, contained at least one binding site with circKEAP1 and signi cantly upregulated in LUAD cancer tissues (Fig. 4B). A dual-luciferase reporter assay was performed to further con rm the interaction between circKEAP1 and its predicted 13 miRNAs in A549 cells. Among the 13 predicted miRNAs, all the miRNAs signi cantly attenuated the luciferase activity of A549 cells (Fig. 4C), and the miR-141-3p was more obvious than other miRNAs and selected for deeper investigation. It has been proved that circRNAs bind with miRNAs through AGO2 (Argonaute 2). Therefore, we conducted the anti-AGO2 RNA immunoprecipitation (RIP) assay in BEAS-2B cells to pull down the RNA transcripts which bind to AGO2 with anti-AGO2 antibody. Interestingly, both circKEAP1 and miR-141-3p were e ciently pulled down by anti-AGO2 antibodies (Fig. 4D). Thus, it's supposed that circKEAP1 might act as a sponge for miR-141-3p. Subsequently, we examined the expression levels of miR-141-3p in the 105 pairs of LUAD cancer tissues and adjacent non-cancerous tissues, and found miR-141-3p was markedly upregulated in LUAD cancer tissues compared with adjacent non-tumor tissues (Fig.4E). As showed in Fig.4F, there are two binding sites between miR-141-3p and circKEAP1 (Fig. 4F). We performed the dual-luciferase reporter assay to con rm the bioinformatics prediction analysis in A549 cells. The full-length of circKEAP1 and muted circKEAP1 with muted miR-141-3p binding sites were subcloned into luciferase reporter pGL3 plasmid. We found miR-141-3p mimic could effectively increase the expression level of miR-141-3p in A549 cells (Fig S1), and signi cantly decreased the luciferase activity of wildtype group, however it had no effect on the mutant group (Fig. 4G). These results suggested there might be a direct interaction between circKEAP1 and miR-141-3p. Then, the miRNA pull-down assay with speci c biotin-labeled miR-141-3p was performed to further verify the binding of circKEAP1 and miR-141-3p. As expected, circKEAP1was signi cantly enriched in the biotin-labeled miR-141-3p group compared with control (Fig. 4H). Additionally, we also found the expression level of miR-141-3p in both A549 cells (Fig. 4I) and tumors ( Fig. S2A) was markedly decreased when we overexpressed the circKEAP1 by circKEAP1 plasmid. The decrease could be attenuated by miR-141-3p mimic in A549 cells (Fig. 4J).
These results reveal that circKEAP1 could server as a sponge for miR-141-3p in LUAD cancer cells.

miR-141-3p repress the KEAP1 expression in tumors
It's well known that circRNAs can act as miRNAs sponge to regulate downstream targets 5, 6 , by sharing the same miRNAs with mRNA 7,19 . Three bioinformatics software TargetScan 18 , miRDB 20 and miRanda 21 were used to predict the target genes of miR-141-3p. Bioinformatics analysis showed that the 3′-UTRs of ZEB1, ZEB2, and KEAP1 contained miR-141-3p complementary sequences by all the three algorithm (Fig. 5A). In order to con rm the repression of miR-141-3p, we rstly performed dual luciferase reporter assay in A549 cells. We found miR-141-3p remarkably suppressed the luciferase activity of luciferase reporter vector containing the KEAP1 3'UTR sequence, while it has no effect on ZEB1 and ZEB2 in A549 cells (Fig. 5B). As predicted, miR-141-3p have one binding sites in the 3'-UTR of KEAP1 (Fig.5C). In order to further con rm the repression of miR-141-3p on KEAP1, we rstly performed dual luciferase reporter assay in A549 cells. We found miR-141-3p remarkably suppressed the luciferase activity of luciferase reporter vector containing the KEAP1 3'UTR wild type sequence (Fig. 5D), while had no in uence on the muted vector containing the muted KEAP1 3'UTR sequence (Fig. 5D). Then, we performed the miRNA pulldown assay with speci c biotin-labeled miR-141-3p, and the result showed KEAP1 mRNA was also speci c enrichment in the biotin-labeled miR-141-3p group, like circKEAP1 (Fig. 5E). The IHC staining for KEAP1 protein in 105 paired LUAD cancer tissues and adjacent normal tissues and western blotting for KEAP1 protein in 12 paired LUAD cancer tissues and adjacent normal tissues was performed to analysis the protein level of KEAP1. The results showed the protein level of KEAP1 was remarkably downregulated in LUAD cancer tissues, compared to the adjacent non-cancerous tissues (Fig. 5F and G). Pearson correlation analysis revealed a signi cant negative correlation between KEAP1 protein level and miR-141-3p. These results suggested miR-141-3p might regulat the protein level of KEAP1. To determine whether miR-141-3p could inhibit KEAP1 expression, BEAS-2B cells were transfected with mimics of miR-141-3p to increase the level of miR-141-3p, while A549 cells were transfected with inhibitors of miR-141-3p to downregulate the cellular levels of miR-141-3p. As anticipated, mimics of miR-141-3p could inhibit KEAP1 expression, while inhibitors increased KEAP1 levels (Fig. 5H). KEAP1 is con rmed to bind to NRF2 (nuclear factor erythroid 2-related factor 2) and promote its degradation by the ubiquitin proteasome pathway, which could in turn repress the HDAC4 (histone deacetylase 4) expression in LUAD 22 .We found miR-141-3p mimics signi cantly increased the NRF2 and HDAC4 expression by repressing the KEAP1 expression, and miR-141-3p inhibitors remarkably suppressed the NRF2 and HDAC4 expression by reliving the suppression of miR-141-3p for KEAP1.
Actually, KEAP1 has been proved to be a target gene of miR-141-3p in LUAD by many previous studies 23,24,25,26,27,28 . Our results con rmed again that miR-141-3p can inhibit KEAP1 by binding to its 3'-UTR. circKEAP1 relieved repression of miR-141-3p for KEAP1 expression To con rm circKEAP1 could regulate the repression of miR-141-3p for KEAP1, the luciferase reporters containing KEAP1 3'UTR wild type sequence or muted sequence was co-transfected with circKEAP1 plasmid or miR-141-3p mimic in A549 cells. The results showed circKEAP1 signi cantly increased the luciferase activity of KEAP1 wild type reporter, while it had no effect on the mutated reporter. Moreover, the increase by the circKEAP1 could be abolished by miR-141-3p overexpression through miR-141-3p mimic (Fig. 6A). Subsequently, the protein level of KEAP1 was evaluated in cells with overexpressing circKEAP1, and resulting to decrease the NRF2 and HDAC4 expression, which was reported as the downstream genes of KEAP1 pathway 29 , but it was rescued by miR-141-3p overexpression (Fig. 6B-C). Additionally, we also found the protein level of KEAP1 was signi cantly upregulated in the tumor of circKEAP1 overexpression group, which in turn downregulated the protein level of NRF2 and HDAC4 (Fig.  S2B).
Several studies had been con rmed KEAP1 could suppress the expression of NRF2, and reduce its downstream gene HDAC4 expression, result to increase the tumor suppressor miRNAs miR-1/206 level to regulates glucose metabolism in cancer cells 29 . In our study, we found circKEAP1 could signi cantly increase the expression of miR-1 and miR-206 both in vitro (Fig. 7A) and in vivo (Fig. S2C), however this increase could be attenuated by miR-141-3p overexpression. Additionally, in accordance with the results before, the circKEAP1 signi cantly repressed the cancer cells proliferation (Fig. 7B-D), and the suppression could be rescued by miR-141-3p overexpression using miR-141-3p mimic (Fig. 7B-D).
Taken together, our results found circKEAP1 could server as a sponge for miR-141-3p to regulate KEAP1 and activate the KEAP1/NRF2/HDAC4 signal pathway via the ceRNA mechanism to repress tumor growth (Fig. 8).

Discussion
Recently, an increasing number of studies have con rmed the dysregulation of circRNAs play critical roles in modulating tumor development and progression 6 . Several circRNA has been revealed to function as oncogenes or tumor suppressors in lung cancer and other types of cancer 6,11,14 . However, only a few circRNAs have been well characterized. In this study, the pro ling of circRNAs in LUAD was obtained by the Agilent-069978 Arraystar Human CircRNA microarray V1. We found circKEAP1 signi cantly downregulated in LUAD cancer tissues, and could suppress the tumor growth. CircKEAP1 exerted its function as a ceRNA that competitively bound to miR-141-3p, then abolished the repression of miR-141-3p for its original gene KEAP1. Elevated KEAP1 could inhibit the expression of NRF2 and HDAC4, and then abolish the suppression of HDAC4 for the tumor suppressor miRNAs miR-1 and miR-206, which result to repress the cell proliferation (Fig. 8). Our results suggested circKEAP1 could inhibit LUAD cell growth via the ceRNA mechanism.
Heterogeneous genetic or epigenetic modi cations have been revealed could modify the oncogenes and tumor suppressor genes expression. The dysregulation of these genes exert their effects on multiple cellular processes in which transient modi cations of redox balance might occur, such as cell proliferation. These transient cellular changes are mainly coordinated by KEAP1/NRF2 signaling pathway 22 . NRF2 is a transcription factor and could function as a master modulator of cellular defense against toxic and oxidative damage, mitochondrial physiology, differentiation, and stem cell maintenance 30 .
NRF2 is tightly regulated by KEAP1 22,30 . In normal cell conditions, KEAP1 forms an ubiquitin ligase complex and targets NRF2 for proteolysis. Upon stress exposure, the KEAP1 releases NRF2 which translocates into the nucleus to form a heterodimeric complex which could recognizes the enhancer sequences of antioxidant response elements (AREs) and activates their transcription 22,30 . Since KEAP1 and NRF2 could modulate cell proliferation, it's considered a hallmark in cancer cells of the deregulation of the KEAP1/NRF2 axis. Previous studies have indicated that abnormal states of the KEAP1-NRF2 pathway exist in lung cancer 30,31 . Downregulated KEAP1 has been frequently identi ed in lung cancer, however the mechanism still remains unknown 31 . In this study, we also found KEAP1 was signi cantly downregulated in LUAD cancer tissues and knockdown of KEAP1 enhance the NRF2 expression. More interestingly, we revealed KEAP1 could encode a circRNA by alternative splicing to sponge miR-141-3p to relive the inhibitor of the miRNA for itself mRNA. In tumor condition, the decrease of the circKEAP1 resulted to the evaluated of miR-141-3p, which in turn suppress the protein level of KEAP1 and increase the NRF2 level. It's reported KEAP1 could modulate the NRF2 to repress HDAC4 methylated the promoter of tumor suppressor miRNA miR-1 and miR-206 29 . In our study, we found circKEAP1 could upregulate miR-1 and miR-206 by upregulating KEAP1, which could decrease NRF2 expression, and result to suppress the HDAC4 and in turn inhibit the methylation of the promoter of these two miRNAs by HDAC4 (Fig. 8).
The most signi cant characteristic of cancer cells is uncontrolled cell proliferation. Previous studies have demonstrated that miR-141-3p was upregulated in multiple human cancers, and high expression of miR-141-3p promoted cancer cell proliferation via varying mechanisms 32 23 . In this study, we found circKEAP1 could inhibit cell proliferation by acting as a sponge for the miR-141-3p to relieve microRNA repression for target gene KEAP1.
In conclusion, our study reveals that circKEAP1 competitively binds miR-141-3p to abolish the suppressive effect of miR-141-3p on KEAP1, which result to suppress the NRF2/HDAC4 signal pathway and inhibit the cell proliferation. Our ndings revealed an insight into understanding the KEAP1-NRF2 signal pathway during the development and progression of LUAD, and provide a potential therapeutic approach for LUAD.