MiR-23a-mediated inhibition of topoisomerase 1 expression potentiates cell response to etoposide in human hepatocellular carcinoma
© Wang et al.; licensee BioMed Central Ltd. 2013
Received: 17 December 2012
Accepted: 2 October 2013
Published: 8 October 2013
microRNAs have been shown to regulate the chemosensitivity of cancer cells. The aim of this study is to investigate the role and mechanism of mir-23a in enhancing the anti-tumor effect of topoisomerase 2A (TOP2A) poison etoposide in human hepatocellular carcinoma (HCC).
The anti-tumor effect of chemotherapeutic agents in HCC cells were examined in vitro and in vivo xenograft model. Expression of mRNA and miRNAs were determined by quantitative real-time PCR. Protein expression was analyzed by immunoblotting.
Overexpression of mir-23a could significantly potentiate the in vitro and in vivo anti-tumor effect of etoposide; however, ectopic expression of miR-23a fails to sensitize HCC cells to 5-fluorouracil treatment, indicating the miR-23a-induced cancer cell hypersensitivity in chemotherapy is TOP2A-specific though miR-23a overexpression could not directly up-regulate TOP2A expression. Topoisomerase 1(TOP1) is down-regulated in miR-23a-overexpressed HCC cells. MiR-23a could directly bind to 3′untranslated region of TOP1 mRNA, and suppress the corresponding protein expression and inhibition of miR-23a further arguments the expression of TOP1. MiR-23a was up-regulated during DNA damage in cancer cells in line with the p53 expression. Up-regulation of p53 induces mir-23a expression, while suppression of p53 inhibits miR-23a in HCC cells.
Our study sheds light on the role of miR-23a as a potential target in regulating chemosensitivity of HCC cells.
MicroRNAs (miRNAs) are small non-coding RNAs with an average length of 20–22 nucleotides. Recent studies have revealed that miRNAs are involved in many different physiological and pathological processes via regulating functional genes’ expression . With direct binding to the 3′-untranslated region (3′-UTR) of these mRNAs, one individual miRNA could regulate hundreds of genes via inducing mRNA degradation or prohibiting gene translation [2, 3]. Accumulating evidences have shown that expression profile of miRNAs differs from that in normal human tissue . The differential expression of miRNAs in consequence regulates oncogenic factors or tumor suppressors, leading to onset/offset of angiogenesis, growth and metastasis of human cancers .
It was further noticed that miRNAs play critical roles in regulating tumor cell response to chemotherapeutic agents. Previous studies have shown that the expression profile of miRNAs in chemoresistant human cancer cell lines varies from chemo-responsive tumor cells . Altered expression of miRNAs could control the chemosensitivity of cancer cells by, either directly regulating apoptosis-related Bcl-2 family proteins to modify the cellular response to apoptosis initiated by chemotherapeutic agents, or modulating drug availability to influence responsiveness of tumor cells indirectly . TOP2A is one of cellular topoisomerases that determines tumor cell response to chemotherapeutics. A recent study found that chemosensitivity of tumor cells could be determined by intracellular topoisomerase level , which provides a novel strategy in enhancing drug response in cancer therapy. Discovered as a Topoisomerase 2A (TOP2A) poison, etoposide is now a frontline chemotherapeutics in treating various human cancers . However, the mechanism of different responses of tumor cells to etoposide is not yet clear. A recent study shows that expression of miRNAs varies in etoposide resistant breast cancer cells , but how miRNAs regulate cell response upon etoposide remains unclear.
In this study, we report that overexpression of miR-23a could sensitize HCC to the in vitro and in vivo treatment of etoposide. MiR-23a fails to boost up response of HCC cells to 5-fluorouracil (5-Fu) treatment, indicating that the regulation of miR-23a on response of HCC cell may be TOP2A poisons-specific. Overexpression of miR-23a could further impair the cell progression through S phase when HCC cells were exposed to etoposide, while the TOP2A expression has not changed. The other topoisomerase, TOP1 was suppressed in miR-23a-overxpressed HCC cells and was validated as the direct target of miR-23a. Suppression of TOP1 expression by miR-23a results in reduction of overall intracellular topoisomerase activity when the cells are exposed to etoposide, which in consequence enhances drug response of HCC cells. Forced overexpression of miR-23a in HCC cells reduces the cytotoxicity of TOP1 poison irinotecan, and up-regulation of miR-23a could be observed in HCC cells upon DNA damage, during which miR-23a may play a role in the intracellular TOP1 homeostasis. Furthermore, we found expression of miR-23a is regulated by p53 in HCC cells. Our findings shed light on the role of miR-23a as a potential target in regulating drug responses of HCC cells.
Materials and methods
Plasmids and siRNA
The pCMV-MIR Vectors with and without human miR-23a expression, luciferase expressing-pMir-Target Vectors with and without TOP1 3′UTR expression were purchased from Origene (USA). The pRL-CMV Renilla Luciferase (Ruc) Control Reporter Vectors were obtained from Promega (USA). The siRNA against human p53 were from Santa Cruz (USA). The scramble negative control to miRNAs (scr negative control) and inhibitor against miR-23a were purchased from Exiqon (Denmark).
Cell line and cell culture
The human hepatocellular carcinoma cells HepG2 and embryonic kidney cell line HEK293T were obtained from American Type Culture Collection (ATCC, USA). MHCC97L cell line was kindly gifted by Dr. Man Kwan from Department of Surgery, The University of Hong Kong and has been used in our previous published studies [11, 12]. Cells were cultured in High Glucose Dulbecco’s Modified Eagle Medium supplemented with 10% Fetal Bovine Serum and 1% Penicillin/Streptomycin.
Cell viability assays
The cell viability was determined by MTT assay. Briefly, cells were seeded into 96-well cell culture plate and received treatments. 4 h before the end of experiment, 10 μl of 3- (4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (5 mg/mL) was added and cells were incubated at 37°C. Then medium was removed and the residue was dissolved in DMSO. The absorbance of each well was read at 570 nm with a microplate reader.
Cells were exposed to UV-C irradiation (254 nm) for 10 min then incubated at 37°C, 5% CO2.
Quantitative real-time PCR
Detection of mRNA Total RNA was extracted with Total RNA purification kit (Norgan, Canada). The Taqman® Gene Expression Assay (Hs00243257_m1) was conducted for the detection of TOP1 mRNA transcripts with LigherCycler480 (Roche, USA). GAPDH (Hs02758991_g1) was used as the loading control. For detection of TOP2A, SYBR Green I qPCR assay was conducted with primers (left: GCGAGTGTGCTGGTCACTAA; right: ACAATTGGCCGCTAAACTTG). GAPDH was used as the loading control (left: GAGTCCACTGGCGTCTTCAC; right: TTCACACCCATGACGAACAT).
Detection of miRNAs The total RNA containing miRNAs were extracted with miRNeasy mini Kit (Qiagen, Germany) under the manufacturer’s instruction. The Taqman® MicroRNA Assay (000399) was conducted for the detection of mature miR-23a with U6 as loading control (001973); The Taqman® Pri-miRNA Assay (Hs04270764_pri) was conducted to detect the expression of pri-miR-23a with GAPDH as loading control (Hs02758991_g1); For the detection of pre-miR-23a, SYBR Green I qPCR Assay was conducted with primers (left: CTGGGGATGGGATTTGCT; right: TGGAAATCCCTGGCAATG). GAPDH was used as the loading control (left: GAGTCCACTGGCGTCTTCAC; right: TTCACACCCATGACGAACAT).
Cells were lysed with Radioimmunoprecipitation assay (RIPA) buffer with complex cocktailed proteinase inhibitor (Roche, USA) and phosphatase inhibitor (1 mM Na3VO4 and 1mM NaF) on ice for 30 minutes followed by centrifugation at 14,000 rpm at 4°C for 15 minutes. Supernatants were transferred and protein concentrations were determined by BSA assay (Bio-rad, USA). Equal yields of protein were separated on SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (PVDF, Biorad). The membrane was then blocked in buffer containing 5% BSA, Tris (10 mmol/L, pH7.4), NaCl (150 mmol/L) and Tween 20 (1%) at room temperature for one hour with gentle shaking. The membrane was then incubated with primary antibodies at 4°C overnight followed by incubation with appropriate secondary antibody (Abcam, UK) at room temperature for 1 hour. The immunoreactivites were detected using ECL advanced kit (GE Healthcare, UK) and visualized using a chemiluminenescence imaging system (Bio-Rad, USA).
Cell cycle analysis
Cells were seeded in 6-well plate with 50% confluence and were synchronized by overnight starvation. After treatment the cells were collected and fixed in 75% alcohol for 24 h. Then cell pellet was stained with 50 μg/mL Propidium iodide (PI) in PBS for 40 min in dark at room temperature. Cells were washed and resuspended and the cell cycle distribution was determined with Flow cytometer (BD FACSCantoII Analyzer, USA). The raw data was collected and analyzed by Flow Jo 7.6 software (USA).
The HEK293T cells were co-transfected with pMIR Vectors containing TOP1 3′UTR and miR-23a mimics. Cells were lysed and the Firefly luciferase activity was detected and Renilla lucfierase activity was used for normalization. The lysate was detected with Dual Luciferase reporter assay kit (Promega, USA) with a luminometer.
The tumor xenograft model was used in this study. Briefly, 6-week-old female BALB/c nu/nu athymic nude mice received MHCC97L cells transfected with pCMV vector injection subcutaneously in 0.2 ml at its left side of waist and MHCC97L cells with miR-23a overexpression injection at its right. Mice was then randomized into different groups (n = 5). Etoposide treatment group received 0.2 mL i.p. injection of 7.5 mg/kg etoposide, while 5-Fu treatment group received the same volume of 25 mg/kg 5-Fu. Control group received the same volume of PBS. Tumor volume and body weight were measured 3 times per week for 4 weeks. At the end of the experiment, the mice were sacrificed with overdose of Phenobarbital (200 mg/kg) and the tumor was dissected out. All animals received human care and study protocols complied with the guidelines of the Laboratory Animal Centre of The University of Hong Kong and were approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR) of the University of Hong Kong. Moreover, animals were processed according to the suggested international ethical guidelines for the care of laboratory animals throughout the experiments.
Results were analyzed using student T-test and expressed as mean ± SD.
Overexpression of miR-23a sensitizes tumor cell to TOP2A poisons
TOP2A is responsible for the hypersensitivity of miR-23a-overxpressing HCC cells to etoposide
TOP1 is the direct target of miR-23a in hepatocellular carcinoma
DNA damage-induced miR-23a regulates TOP1 expression in hepatoceullar carcinoma cells
Expression of miR-23a was transcriptional activated by p53
The role of miR-23a in tumor progression is far from conclusive and still under investigation. Some previous studies showed that miR-23a could function to promote tumor progression by inducing tumor cell proliferation and invasion [3, 23]. In our study, we found that expression of miR-23a may increase in HCC tissue when compared with non-tumor livers, which is consistent with some previous reports . Mir-23a was showed to regulate the glucose metabolism during tumorigenesis via suppressing gluconeogenesis related genes. High levels of aerobic glycolysis are therefore induced, which lead to cancerous transformation of normal cells . Moreover in our study, we found that miR-23a could target TOP1, which is essential to maintain genomic stability during DNA damage . Loss of genomic stability once TOP1 is suppressed may therefore drive cancer development. However, as it was found that TOP1/TOP2A expression in cancer cells are important in determining the cellular response to chemotherapeutics. It was recently observed that inhibition of TOP1 level could potentiate response of tumor cells to doxorubicin treatment without altering the expression level of TOP2A . Although the underlying mechanism remains not clear yet, it was shown in yeast that overall amount of topoisomerase activity is necessary for cell survival since more severe deficiencies of DNA unwinding, cell cycle progression and chromatin structure could be found in TOP1/TOP2A double mutant strains in compared to each single mutant [25, 26]. It was therefore suggested that cells are vulnerable if the overall activity of topoisomerases is below the minimal level that cell itself sets . Consistently it was observed in our study that TOP1 down-regulation by miR-23a in HCC cells could largely potentiate cells to etoposide and doxorubicin treatment but not 5-Fu exposure, and forced overexpression of miR-23a in HCC cells reduced the cytotoxicity of TOP1 poison irinotecan, whose activity was dominated by the content of endogenous TOP1 level . The simultaneous TOP2A poisoning by etoposide with TOP1 inhibition results in cellular topoisomerase activity falling below the crucial threshold and triggering death of the cell. As etoposide is a TOP2A poison that induce covalent binding of TOP2A protein with DNA and results in DNA break and fragment in cancer cells, the etoposide sensitivity may be associated with the DNA repair mechanism in which the poly (ADP-ribose)polymerase (PARP) cleavage may be involved. Indeed, it was found that in etoposide-resistant cancer cells, deficiency of PARP cleavage was observed upon etoposide treatment . It is probable that the compensatory activation of PARP cleavage may be initiated when TOP1 activity has also been suppressed in etoposide-treated cancer cells since previous study has found PARP cleavage in cells with TOP1 being inhibited . As cleavage of PARP eventually results in cancer cell death, this compensatory action of TOP1 reduction in etoposide-treated cancer cell may therefore increase the chemotherapeutic sensitivity.
In closing, overexpression of miR-23a potentiates HCC cell to etoposide-induced cell death. Overexpression of miR-23a could not synergize 5-Fu-induced cytotoxicity and tumor inhibition, which indicates the mechanism of hypersensitivity induced by miR-23a is TOP2A poison-specific. Although TOP2A expression remained unchanged in miR-23a-overexpressing HCC cells, TOP1 was remarkably down-regulated, which may lead to the overall topoisomerase activity fall below the critical threshold for cell survival when cells are exposed to TOP2A poisons and consequently accelerates cell death. Both mRNA transcripts and protein expression of TOP1 are suppressed in miR-23a-overexpressing HCC cells, and luciferase assay shows that miR-23a may directly bind to the 3′UTR of TOP1 mRNA to suppress its expression. The interaction between miR-23a and TOP1 was further evidenced by the fact that forced overexpression of miR-23a could attenuate the cytotoxicity of TOP1 poison irinotecan. Activation of miR-23a could be induced during DNA damage in parallel with up-regulation of p53 expression. Activation of p53 could increase the transcripts of pri-, pre- and mature form of miR-23a, while inhibition of p53 significantly reduced miR-23a level in p53-proficient HCC cells, indicating that miR-23a may be transactivated by p53 in HCC cells. Our study shed lights on the potential of miR-23a as a novel target in regulating chemosensitivity of cancer cells.
The study was financially supported by grants from the research council of the University of Hong Kong (Project Code: 10401764), The Research Grant Committee (RGC) of Hong Kong SAR of China (RGC General Research Fund (Project Code: 10500362). The authors would like to express thanks to Ms Oi Yee Chow, Ms Cindy Lee, Mr. Keith Wong, and Mr. Freddy Tsang for their technical support. The author acknowledges the assistance of the University of Hong Kong, Li Ka Shing Faculty of Medicine Faculty Core Facilities.
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