ANRIL promotes chemoresistance via disturbing expression of ABCC1 by regulating the expression of Let-7a in colorectal cancer

Increasing evidence indicates that long non-coding RNAs (lncRNAs) antisense non-coding RNA in the INK4 locus (ANRIL) has been involved in various diseases and promotes tumorigenesis and cancer progression as an oncogenic gene. However, the effect of ANRIL on chemoresistance remains still unknown in colorectal cancer (CRC). Here, we investigated ANRIL expression in 63 cases of colorectal cancer specimens and matched normal tissues. Results revealed that ANRIL was up-regulated in tumor tissues samples from patients with CRC and CRC cell lines. Increased ANRIL expression in CRC was associated with poor clinical prognosis. Kaplan–Meier analysis showed that ANRIL was associated with overall survival of patients with colorectal cancer, and patients with high ANRIL expression tended to have unfavorable outcome. In vitro experiments revealed that ANRIL knockdown significantly inhibited CRC cell proliferation, improved the sensitivity of chemotherapy and promoted apoptosis. Further functional assays indicated that ANRIL overexpression significantly promoted cell chemoresistance by regulating ATP-binding cassette subfamily C member 1 through binding Let-7a. Taken together, our study demonstrates that ANRIL could act as a functional oncogene in CRC, as well as a potential therapeutic target to inhibit CRC chemoresistance.


Introduction
Colorectal cancer (CRC) is the third most common human cancer and the fourth most common cause of cancer deaths in global and its incidence has increased in recent years [1]. Although encouraging advances in diagnosis and cancer therapy in the past decade, CRC remains a high-risk digestive tract tumor with the low overall survival rate due to CRC metastasis and chemoresistance. Moreover, the prognosis and treatment strategies of CRC rely on tumor stage [2]. Compared with higher 5-year survival rate for patients with early stage, the 5-year survival for patients with advanced stage is lower [3,4]. Despite receiving standard chemotherapy regimens, most of CRC patients eventually generate chemoresistance and cause to treatment failure [5]. Even though great efforts have spent to explore the cause of CRC tolerance for chemotherapy, the molecular mechanisms of CRC chemoresistance remain unclear. Revealing the underlying mechanisms of chemoresistance is now essential to overcoming chemoresistance of patients who receive systemic therapy with CRC.
Chemotherapy is a common and effective method to treat tumor. 5-Fluorouracil (5-FU) is one of the effective and commonly used chemotherapeutic agents for CRC and other tumors including pancreatic cancer, esophageal cancer, gastric cancer, liver and breast cancers. It disturbs DNA replication by replacing thymidine with fluorinated nucleotides that are incorporated into DNA thus causing cell death [6].
In clinical, it often treats CRC alone or in combination with cetuximab, oxaliplatin and leucovorin for increasing response rates of cancer cells [7,8]. Although diverse approaches have been taken consideration into solving chemoresistance to 5-FU, resistant to 5-FU-based chemotherapy is a major hindrance to effective chemotherapy in CRC and others yet.
In the recent decades, numerous long non-coding RNAs (lncRNAs) are continuously discovered and simply classified as transcripts longer than 200 nucleotides with unapparent coding potential to be similar with most mRNAs [9]. Contrast to initial understanding for lncRNAs as 'transcriptional noise' , increasing evidence shows that lncRNAs play important regulatory role in biological processes and diverse diseases [10]. Moreover, emerging evidence shows that lncRNAs have been identified to be closely interrelated with chemoresistance of human cancers. An oncogene HOTTIP enhances chemoresistance of SCLC through regulating BCL-2 by binding miR-216a in SCLC progression [11]. LncRNA LINC00161 increases cisplatin-induced apoptosis and reverses the cisplatin resistant phenotype of osteosarcoma cells through up-regulated expression of IFIT2 by sponging endogenous miR-645 [12]. The antisense non-coding RNA in the INK4 locus (ANRIL) is transcribed as 3834-nt lncRNA regarded as a risk factor in tumorigenesis [13]. For example, overexpression of ANRIL is involved in repressing of the p16/ARF gene cluster in cis by directly binding to the PRC1 via CBX7 in prostate cancer [14]. ANRIL can recruit PRC2 to the P15 promoter and repress the expression of P15 [15]. Previous studies have reported that ANRIL was associated with the survival rate of CRC patients and promoted cell migration and invasion, promoted lymphangiogenesis as well as lymphatic metastasis [16,17]. Nevertheless, the effect of ANRIL on resistance to CRC chemotherapy remains unknown. Therefore, we hypothesize that the ANRIL may be involved in CRC chemoresistance.
Here, our study shows that ANRIL is overexpressed in CRC tissues and CRC-derived cell lines. We find overexpression of ANRIL promotes significantly CRC cell migration and drug resistance. Furthermore, the mechanism study reveals that ANRIL promoted the expression of ATP-binding cassette subfamily C member 1 (ABCC1) by regulating Let-7a. Our results provide novel insights into the function of ANRIL in CRC chemoresistance.

Tissue samples
All of 63 CRC specimens and adjacent non-neoplastic tissue were collected from each of the patients between 2013 and 2017 at Yan'an University Affiliated Hospital (Yanan, China). The Institutional Review Board Affiliated to Yan'an University Affiliated Hospital (Yanan, China) approved the protocol of the present study to be used for the patients at Yan'an University Affiliated Hospital, and informed consent was collected from each patient prior to surgery. All CRC patients did not receive any treatments including chemotherapy, radiotherapy or any other medical intervention prior to the surgery. These tissue specimens were frozen in liquid nitrogen and stored at −80 • C. All tissues were pathologically confirmed according to the World Health Organization (WHO) classifications and collected before chemotherapy. Tumor response was confirmed through computed tomography and evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST) criteria as complete response (CR), partial response (PR), stable disease (SD) and progressive disease (PD).

Ethics statement
The Institutional Review Board Affiliated to Yan'an University Affiliated Hospital (Yanan, China) approved the protocol of the present study to be used for the patients at Yan'an University Affiliated Hospital, and informed consents were collected from each patient prior to surgery. All experiments were performed in accordance with relevant regulations and guidelines.

Cell lines and cell culture
A human fetal normal colonic cell FHC and the CRC cell lines LOVO, HCT116, HT29, RKO, SW480 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cells cultured in RPMI1640 (Life Technologies, U.S.A.) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin (Life Technologies, U.S.A.) in humidified air at 37 • C with 5% CO 2 .

RNA extraction and qRT-PCR
Total RNA was extracted from tissues or cells using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The condition of RNA reverse transcription to cDNA was performed with 42 • C for 5 min, 99 • C for 20 min and 4 • C for 5 min by a Reverse Transcription Kit (Takara). Quantitative real-time PCR (qRT-PCR) analyses used SYBR Green I (Bio RAD) in triplicate. The results were normalized to the expression of GAPDH or U6. All primer sequences used for qRT-PCR were listed in Supplementary Table S1.

Colony formation, Transwell assays and wound healing assay
Cells transfected with si-NC, si-ANRIL, pcDNA3.1 vector or pcDNA3.1-ANRIL were harvested 48 h after transfection by trypsinization and seeded into six-well plates (2000 cells per well) for culturing 2 weeks. Visible colonies were fixed with methanol and stained with 0.1% crystal violet. Colonies with more than 50 cells were counted. For Transwell assays, cells transfected with si-NC, si-ANRIL, pcDNA3.1 vector or pcDNA3.1-ANRIL were harvested 48 h after transfection by trypsinization and seeded into the upper chamber 3 × 10 4 /100 μl, the lower chambers of the Transwell were filled with 500 μl RPMI1640 containing 10% FBS and incubated for 24 h at 37 • C in an atmosphere containing 5% CO 2 . Following 24 h incubation, the cells located on the lower surface of the chamber were stained and counted using a microscope (Nikon, Tokyo, Japan). For wound healing assay, the transfected CRC cells (1 × 10 6 /well) were grown to 90% confluence in six-well plates. Cell monolayers were then scratched by a 200-μl sterile pipette tip and washed gently with PBS three times. Then, fresh medium was replenished for an additional incubation of 24 h. The wound areas were imaged at 0 and 24 h with an inverted microscope (Nikon, Tokyo, Japan).

Luciferase reporter assays
To detect the interactions between Lnc RNA ANRIL and Let-7, Let-7a and ABCC1, a dual luciferase reporter assay was performed. HCT116 cells were seeded at 4 × 10 4 cells per well in 24-well plates, and 24 h after plating, the cells were cotransfected with the dual luciferase psiCHECK-2 wild-type/mutant-type (100 ng) (Generay Biotechnology, Shanghai, China) and mimics/control or (50 nmol/l) using Lipofectamine TM 2000 (Invitrogen, U.S.A.) according to the manufacturer's instructions. The cells were mixed well with PLB buffer from the dual-luciferase reporter assay system (Promega, Madison, WI). The Renilla luciferase signal was normalized to the firefly luciferase signal.

Flow cytometry
Cell apoptosis and cell cycle assays were determined with Annexin V-FITC and propidium iodide (PI) by flow cytometry analysis. Briefly, cells were treated with or without 5-FU, cetuximab or oxaliplatin for 24 h after transfection. Cells were harvested by trypsinization and centrifuged to remove the medium, washed once with binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl 2 in aquadest) stained with FITC-Annexin V and PI (BD, San Diego, CA, U.S.A.) according to the manufacturer's instructions. Cells were detected by flow cytometry (FACScan; Becton Dickinson, MountainView, CA, U.S.A.). Viable cells were negative for both PI and AnnexinV, while apoptotic cells were positive for Annexin V and negative for PI; late apoptotic dead cells displayed both high labeling Annexin V and PI. Nonviable cells that underwent necrosis were positive for PI and negative for AnnexinV. Apoptotic rate was compared with control treatment from each experiment.

Statistical analysis
Statistical analyses were performed using Graph Pad Prism (San Diego, CA). Data are presented as the means + − SD of at least three independent experiments. The differences between groups were tested using a two-tailed Student's t-test. Relationships between ANRIL expression and clinic pathologic characteristics were determined by the χ 2 test. Pearson's correlation coefficient was used to measure the linear relationship between the expression levels of ANRIL and Let-7a in CRC tissues. Differences were considered significant at P<0.05.

ANRIL is up-regulated in colorectal cancer tissues and cells
To investigate the effect of ANRIL on the progression of CRC, the expression levels of ANRIL were detected in 63 paired CRC tissues and adjacent normal tissues by qRT-PCR. The expression of ANRIL was significantly increased in 63 CRC tissues compared with paired non-cancerous tissues ( Figure 1A). Moreover, the CRC tissues in 68.3% (43 of 63) of cases had at least two-fold higher expression of ANRIL than paired non-cancerous tissues ( Figure 1B). We further evaluated the expression levels of ANRIL in the five CRC cell lines LOVO, RKO, HT29, SW480 and HCT116. The expression levels of ANRIL were also significantly up-regulated in these CRC cell lines compared with FHC normal colorectal epithelium cells ( Figure 1C). Furthermore, to investigate the clinical prognosis significance of ANIRL, ANRIL expression was divided into a high-expression group (n=43) and a low-expression group (n=20) by a median ANRIL expression level of 3.264. Overall survival was significantly shorter in the high ANIRL expression group than in the low ANRIL group ( Figure 1D). Furthermore, we found ANRIL expression was correlated with disease stage, the expression of ANRIL was higher in patients with advanced stage or with lymph node metastasis (LNM) compared with patients with stage I-II or without LNM ( Figure 1E,F). These results showed that ANRIL expression was positively associated with poor clinical prognosis in CRC. Meanwhile, we sought to explore the association between ANRIL and response to treatment in samples from patients with CRC treated with 5-FU by using the RT-qPCR-D method. Patients were divided into responding (CR + PR) and non-responding (SD + PD) groups according to RECIST criteria. We found that the ANRIL expression level was much higher in patients who did not respond to treatment than those who experienced response to chemotherapy ( Figure 1G).

ANIRL promotes proliferation and migration of colorectal cancer cell lines in vitro
We next investigated the role of ANRIL in CRC by knockdown expression of ANRIL in CRC cell lines HCT116, HT29 and SW480. We choose si-ANRIL (NO.3), HCT116 and SW480 for further experiments due to higher efficient inhibition of si-ANRIL (Figure 2A). For the cell proliferation assays, we detected the cell viability of HCT116 and SW480 cells with treatment of transfected si-ANRIL or si-NC grown into 96-well plates for indicated time by CCK-8 assay. We found that the decreased expression of ANRIL inhibited the cell proliferation ( Figure 2B,C). Consistent with the results, the colony formation assay showed the same result ( Figure 2G). Meanwhile, wound healing assay ( Figure 2D,E) and Transwell assay ( Figure 2F) shown the migration potential of HCT116 and SW480 with transfected siANRIL was obviously inhibited, compared with the cells with transfected si-NC. Additionally, we observed the IC 50 value of 5-FU and oxaliplatin for si-ANRIL transfected HCT116, and SW480 cells was lower than that for si-NC transfected HCT116 and SW480 cells ( Figure 2H-J). Therefore, these results indicated that the up-regulated expression of ANRIL promoted CRC cells proliferation, metastasis as well as chemoresistance.

Knockdown of ANRIL enhanced colorectal cancer cells sensitive to 5-FU and oxaliplatin
Based on the above results, we next to demonstrate that ANRIL has an important role in resistance to 5-FU and oxaliplatin. We first detected the effect of 5-FU on HCT116 and SW480 transfected with si-ANIRL or si-NC by flow cytometer. 5-FU was added for 48 h at concentration of IC 50 after transfecting with si-ANRIL or si-NC for 24 h. We found that cell apoptosis of treating si-ANRIL-transfected HCT116 and SW480 cells with 5-FU was more than si-NC-transfected cells treated with 5-FU ( Figure 3A-D). These results showed that knockdown of ANRIL enhanced the sensitivity of the CRC cells to 5-FU. Furthermore, the cell viability assays were conducted by CCK-8.
Si-ANRIL-transfected HCT116 and SW480 cells were respectively planted into 96-well plates for incubating indicated time after adding 5-FU and oxaliplatin at concentration of respective IC 50 . We found the cell viability of si-ANRIL-transfected HCT116 and SW480 cells with treated 5-FU or oxaliplatin was significantly declined, compared with si-NC-transfected cells with treated 5-FU or oxaliplatin ( Figure 3E-H). These data indicated ANRIL promoted CRC resistant to chemotherapy.

ANRIL positively regulates ABCC1 expression in CRC cells
To further investigate that the downstream target gene was regulated by ANRIL, we detected the effect of ANRIL knockdown and overexpression on two members of ATP-binding cassette (ABC) transporters protein family by qPCR, ABCC1 and ABCC5, which were closely associated with multidrug resistance [18]. We found the expression of ABCC1 and ABCC5 was decreasing in HCT116 and SW480 cells transfected with si-ANIRL, compared with cells transfected with si-NC ( Figure 4A,B). Moreover, we regulated up the expression of ANRIL by pcDNA-ANRIL ( Figure 4C,D). The expression of ABCC1 and ABCC5 was up-regulated ( Figure 4E,F). We also analyzed the data of TCGA samples from bioinformatics website (UALCAN) [19]. The result shown the expression level of ABCC1 in CRC tumor was higher than that in normal tissues. However, the expression of ABCC5 was not significant difference in CRC tissues compared with normal tissues (Figure 4G-J). To further verify the result, we detected the expression of ABCC1 and ABCC5 in 63 tumor specimens and adjacent non-neoplastic tissues by qPCR. Consistent with above results, ABCC1 expression was higher in tumor samples compared with normal tissues, and ABCC5 expression was not significance ( Figure 5A).

ANRIL interacts with Let-7a that is associated with regulation of ABCC1 in CRC
Based on above results, we used analysis with bioinformatics databases (Starbase, RNAhybrid), a computer algorithm to identify miRNA target genes, including lncRNAs [20] that predict potential interactions of lncRNA and miRNA revealed that Let-7a is a putative ANRIL-binding miRNA ( Figure 5B). Further in a study, we demonstrated the expression of ANRIL was negatively correlated with the expression of Let-7a in CRC tissues ( Figure 5B). Using TargetScan ( http://www.targetscan.org/) and Starbase 2.0 ( http://starbase.sysu.edu.cn/mirLncRNA.php), we identified a Let-7a binding site on ABCC1 ( Figure 5B). We also detected the expression of ABCC1 and Let-7a in tissues samples and analyzed the association of ABCC1 expression and Let-7a expression with overall survival by Kaplan-Meier survival analysis. Results revealed that the expression of ABCC1 in high ANRIL expression group was higher than the low ANRIL expression group (Supplementary Figure S1A). More importantly, the Kaplan-Meier survival analysis showed that patients with low ANRIL and low ABCC1 expression had the longest overall survival, while the high ANRIL and high ABCC1 group showed the shortest overall survival (Supplementary Figure S1B). Meanwhile, the expression of Let-7a in high ANRIL expression group was lower than the low ANRIL expression group ( Supplementary Figure S1C), and the Kaplan-Meier survival analysis showed that patients with low ANRIL and high Let-7a expression had the longest overall survival, while the high ANRIL and low Let-7a group showed the shortest overall survival (Supplementary Figure S1D). Contrast to high expression of ANRIL in CRC cell lines, the expression of Let-7a was and SW480 cells transfected Let-7a negative control (Let-7a NC), Let-7a inhibitor (Let-7a in) and Let-7a mimics (Let-7a mi). (G) The luciferase reporter assay was performed in HCT116 cell transfected ANRIL wild-type/mut and Let-7a mimics/negative control, as well as ABCC1 wild-type/mut and Let-7a mimics/negative control. Data represent three independent experiments (mean + − SD of triplicate samples); *P<0.05, **P<0.01, ***P<0.001.
down-regulated, compared with FHC ( Figure 5C). These results shown ANRIL may regulate the expression of ABCC1 by inhibition of Let-7a. We further demonstrated the relationship between ANRIL and Let-7a by ANRIL knockdown and ANRIL overexpression in HCT116 and SW480 cells ( Figure 5D). For further verify the relationship of regulation between ANRIL and Let-7a, we performed luciferase reported gene assay. Results revealed that the luciferase activity was decreased in HCT116 cotransfected with ANRIL wild and Let-7a mimics ( Figure 5G). These results indicated Let-7a expression was regulated down by ANRIL. In order to investigate the relationship between Let-7a and ABCC1, HCT116 and SW480 cells were grown on 12-well plates for 24 h before transfecting Let-7a inhibitor, Let-7a mimics and Let-7a negative control ( Figure 5E,F). The expression of Let-7a was decreasing in HCT116 and SW480 cells transfected inhibitor and increased by Let-7a mimics. Meanwhile, the expression of ABCC1 in HCT116 and SW480 cells transfected Let-7a inhibitor or mimics was accordingly increasing or decreasing ( Figure 5F).The luciferase reporter gene assay revealed the luciferase activity was decreased in HCT116 transfected with ABCC1 wild and Let-7a mimics, while increased in combination of ABCC1 mut and Let-7a mimics ( Figure 5G). These results suggest that the relationship between Let-7a and ABCC1 was negatively regulating.

Discussion
Recently, the effect of lncRNAs on many diseases including carcinogenesis has gotten a lot of attention, and provided the new underlying molecular mechanisms by which lncRNAs involved in the pathogenesis and development of multiple type of human cancers [21,22]. Increasing studies have verified the association of lncRNAs with tumor growth, epithelial-mesenchymal transition and metastasis [23][24][25]. These results of the present study demonstrated that higher levels of ANRIL are present in CRC tissues compared with adjacent normal tissues. The relatively high expression of ANRIL was significantly associated with decreased overall survival rate and poor clinical prognosis. We also found that the expression of ANRIL in CRC cell lines was significantly up-regulated. In addition, ANRIL gene expression was successfully silenced in human CRC cells. ANRIL knockdown decreased proliferation, inhibited migration and reduced the colony forming ability of CRC cells. Consistent with lots of previous studies [26,27], these results support ANRIL have an important role in CRC as an oncogene.
In clinical, acquired drug resistance and enhanced metastasis frequently follow chemotherapeutic regimens, leading to treatment failure in tumor patients [18]. By using the RT-qPCR-D method [28], we validated that high ANRIL expression was negatively associated with chemotherapy response in CRC patients receiving 5-FU-based chemotherapy. This is also considerable therapeutic significance because of the importance of discovering an ANRIL inhibition method that may provide a new modulation strategy to overcome chemoresistance.
We attempted to unravel the molecular switch of ANRIL controlling this chemoresistance phenotype and elucidate the underlying mechanisms on CRC chemoresistance. In our study, we found ANRIL promoted CRC cells resistant to 5-FU and oxalipaltin. Emerging evidence shows the role of ABC transporters protein family in tumor cells acquired drug resistance. ABCC1 and ABCC5 are two members of ABC transporters protein family, which are tightly linked to the generation of chemoresistance in tumor cells [18]. It's reported that Genetic variability in ABCC1/MRP1 was associated with severe hematological toxicity of FEC [29]. In addition, aberrant NRF2 activation is increased via B-Raf-mediated NRF2 gene transcription and HATs-mediated NRF2 protein acetylation, which increases the acquired resistance and promotes the survival of Top II poison-resistant cancer cells. Increased binding of NRF2 to an ARE in the promoter of ABCC1 directly contributed to Top II poison resistance [30]. In the present study, with high ANRIL expression, the expression of ABCC1 and ABCC5 was increasing. These results indicated the role of ANRIL was important in the regulation of ABCC1 and ABCC5. However, there was no significant difference between the expression of ABCC5 in our CRC specimens and adjacent normal tissues.
We further hypothesized that ANRIL regulated the expression of ABCC1 through miRNA according to numerous evidence about the regulation of lncRNA and proteins. For example, the overexpression of the long non-coding RNA MIR100HG promoted cetuximab-resistant through increased repressed five Wnt/β-catenin negative regulators leading to up-regulated Wnt signaling, in cetuximab-resistant colorectal cancer and head and neck squamous cell cancer cell lines and in tumors from colorectal cancer patients that progressed on cetuximab by regulating the expression of miR-100 and miR-125b [31]. The lncRNA CRNDE could regulate the progression and chemoresistance of CRC via modulating the expression levels of miR-181a-5p and the activity of Wnt/β-catenin signaling [32]. In our study, we found the expression of Let-7a was negatively correlated with the expression of ANRIL in CRC tissues and CRC cell lines by qPCR. In addition, ANRIL up-regulated the expression of ABCC1 by inhibition of Let-7a expression, which was predicted to be associated with ANRIL through bioinformatics website.
In conclusion, we proved that ANRIL expression in clinical colorectal cancer was associated with overall survival, poor prognosis and chemoresistance, indicating that ANRIL may be a mediator for the functions of oncogene in CRC progression. Moreover, our study demonstrated that ANRIL promoted CRC cells proliferation and migration, consistent with the result of previous studies about other tumors [33]. Meanwhile, our study provided the first evidence that ANRIL was closely correlated with chemoresistance in colorectal cancer. ANRIL induced chemoresistance for 5-FU and oxaliplatin through ABCC1 expression by regulating Let-7a. These results support strongly ANRIL that may be a potential predictive marker of prognosis for patients with colorectal cancer and function as a promising chemotherapeutic target.