European Review for Medical and Pharmacological Sciences 2019; 23: 5158-5167 MiR-451a suppresses cell proliferation, metastasis and EMT via targeting YWHAZ in hepatocellular carcinoma G.-Y. WEI1, M. HU2, L. ZHAO1, W.-S. GUO1 1Department of General Surgery, Henan Traditional Chinese Medicine Hospital, Zhengzhou, China 2Department of Infection Management, Henan Traditional Chinese Medicine Hospital, Zhengzhou, China Abstract. – OBJECTIVE: MicroRNAs (miR- lines. Moreover, miR-451a inhibited the prolif- NAs) have been identified to participate in eration, invasion and migration of HCC cells via the progression of hepatocellular carcinoma targeting YWHAZ. Our findings indicated that (HCC). However, the function of miR-451a in miR-451a could serve as a novel target for HCC HCC remains unknown. The aim of this study diagnosis and biological therapy. was to determine the function of miR-451a by construction of several experiments in HCC tis- Key Words: sues and cells. MiR-451a, Hepatocellular carcinoma (HCC), Prolifer- PATIENTS AND METHODS: The expression ation, Metastasis, YWHAZ. level of miR-451a in 69 paired of HCC and ad- jacent normal tissue samples was detected us- ing quantitative Real-time polymerase chain re- Introduction action (qRT-PCR). MiR-451a expression in HCC derived cell lines was detected as well. By trans- fecting with miR-451a mimics or inhibitor, the Hepatocellular carcinoma (HCC) accounts expression level of miR-451a in HepG2 or Huh-7 for 85-90% of primary liver cancer. HCC is the cells was up- or down-regulated. MTT (3-(4,5-di- fifth most common morbidity and third most methylthiazol-2-yl)-2,5-diphenyl tetrazolium bro- common mortality cancer worldwide1. In recent mide) assay and colony formation analysis were years, comprehensive treatment based on surgical employed to evaluate the changes of cell pro- liferation. Cell migration and invasion abilities resection has made great progress. However, the were measured via transwell assay. Meanwhile, overall 5-year survival rate of HCC is far from the underlying mechanism of miR-451a in HCC satisfactory2. Metastasis and recurrence of HCC was demonstrated and verified by dual-lucifer- have become a bottleneck restricting long-term ase assay and Western blotting, respectively. survival of patients3-5. Therefore, it is of great sig- Rescue experiments were used to identify the nificance to explore the molecular mechanism of downstream molecule of miR-451a in HCC. RESULTS: MiR-451a expression in HCC tis- HCC development, invasion and metastasis, and sue samples was significantly lower than that of to find novel therapeutic targets. In recent years, 6 adjacent normal samples. Meanwhile, the level researches have found that microRNAs (miR- of miR-451a in HCC cell lines was significantly NAs) play an important role in the development, down-regulated when compared with normal hu- diagnosis and treatment of various tumors, in- man hepatic cell line LO2. MTT assay and colony cluding HCC. MiRNAs are a class of endogenous, formation analysis showed that over-expressed non-coding, and single-stranded RNAs with 21 to miR-451a remarkably inhibited proliferation of HepG2 cells, whereas down-regulated miR-451a 23 nucleotides in length. It can bind completely promoted growth of Huh-7 cells. Transwell re- or partially to the 3’ non-coding region (3’-UTR) sults indicated that up-regulation of miR-451a of target mRNA molecule. This may result in its significantly decreased HCC cell invasion and cleavage or translational blockade, specific in- migration, while down-regulation remarkably in- hibition of gene expression, and involvement in creased cell metastasis. Furthermore, YWHAZ the regulation of life processes7,8. For instance, was identified as a direct target for miR-451a in HCC cells. down-regulation of miR-194-5p inhibits breast CONCLUSIONS: The expression level of miR- cancer cell proliferation, invasion and migration 9 451a was decreased in HCC tissues and cell via repressing Wnt/β-catenin signaling axis . In 5158 Corresponding Author: Weisheng Guo, MM; e-mail: [email protected] MiR-451a suppresses HCC via YWHAZ human gastric cancer, miR-17-92 acts as an on- (HepG2, Huh-7, Hep3B, and SSMC-7721) were cogene and regulates nuclear factor-kappa B (NF- purchased from Shanghai Cell Bank of the Chi- κB) signaling pathway by targeting TRAF310. nese Academy of Sciences (Shanghai, China). Meanwhile, miR-106b-5p promotes cell growth All cells were cultured in Dulbecco’s Modi- and inhibits cell apoptosis in non-small-cell lung fied Eagle’ Medium (DMEM) medium (Gbico, cancer (NSCLC) through regulating BTG311. Fur- Rockville, MD, USA) containing 10% fetal bo- thermore, miR-101 decreases the proliferation and vine serum (FBS, Gbico, Rockville, MD, USA), metastasis of pancreatic cancer by down-regulat- 100 U/mL penicillin and 100 μg/mL strepto- 12 13 ing STMN1 . Previous works have demonstrat- mycin in a 37°C, 5% CO2 incubator. For trans- ed that miR-451a regulates the growth and metas- fection, miR-451a mimics, miR-451a inhibitor, tasis of multiple tumors through different targets. and pcDNA-YWHAZ plasmid over-express- However, its exact role and function in HCC has ing YWHAZ were supplied by Genephrama not been elucidated. In our study, we first detect Co. (Shanghai, China). Briefly, a total of 1×105 the relative expression of miR-451a in 69 pairs HepG2 or Huh-7 cells were seeded into 6-well of HCC tumor tissues and matched normal tis- plates. MiR-451a mimics or inhibitor was sues, as well as in HCC cell lines. Subsequent- transfected into HepG2 or Huh-7 cells accord- ly, HepG2 and Huh-7 cells were transfected with ing to the instructions of Lipofectamine 3000 miR-451a mimics or inhibitor to over-express kit (Invitrogen, Carlsbad, CA, USA). NC and or knockdown miR-451a. Several functional ex- INC were used as negative control group and periments, including MTT (3-(4,5-dimethylthi- inhibitor negative control group, respectively. azol-2-yl)-2,5-diphenyl tetrazolium bromide) as- Hep G2 cells in logarithmic growth phase were say, colony formation assay and transwell assay transfected with pcDNA-YWHAZ and Lipo- were employed to demonstrate the influence of fectamine 3000. 48 h after transfection, cells miR-451a on cell growth, invasion and migration. were collected for subsequent experiments. Western blotting and dual-luciferase analysis were used to explore the downstream molecules of miR-451a in HCC. Our study demonstrated RNA Extraction and Quantitative that miR-451a acted as a tumor suppressor in Real-Time Polymerase Chain Reaction HCC, and significantly inhibited tumor progres- (qRT-PCR) sion via YWHAZ. Our findings indicated that Total RNA in HCC tissues and experimental miR-451a could be used as a potential target for cells were extracted from using TRIzol reagent HCC biotherapy. (Invitrogen, Carlsbad, CA, USA). Extracted RNA was reverse transcribed into complementary de- oxyribose nucleic acid (cDNA). By using cDNA Patients and Methods as a template, the relative expression of miR-451a was detected by the SYBR Green (TaKaRa, Da- HCC Tissues and Cell Lines lian, China) method according to the instructions. A total of 69 paired HCC tissues and adjacent U6 was used as an internal reference. Relative ex- normal tissues were taken from the specimen li- pression level of miR-451a was calculated by the brary of Henan Traditional Chinese Medicine 2-ΔΔCt method. Hospital. Patients received no radiotherapy, che- motherapy or immunotherapy before or during MTT Assay surgery. The collection of HCC tissue specimens Cells transfected with miR-451a mimics or in- was approved by the Ethics Committee of Henan hibitor were first seeded into 96-well plates, fol- Traditional Chinese Medicine Hospital. Informed lowed by culture in a 37°C, 5% CO2 incubator for consent was obtained from each patient before 1, 2, 3, and 4 day, respectively. 20 μL of MTT the study. All specimens were washed with ster- solution (5 mg/mL) (Beyotime, Shanghai, China) ile and non-enzymatic water within 30 min after were added to each well, and cultured for 4 h. Af- excision, and then placed in liquid nitrogen for ter discarding the old culture solution, 150 μL of subsequent use. dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) were added to each well and Cell Culture and Transfection shaken at room temperature for 10 min. Optical HEK 293T cell line, human normal hepatic density (OD) value at the wavelength of 490 nm cell line (LO2), and human hepatoma cell lines was measured by a microplate reader. 5159 G.-Y. Wei, M. Hu, L. Zhao, W.-S. Guo Colony Formation Analysis were separated by 10% sodium dodecyl sul- Cells transfected with miR-451a mimics or in- phate-polyacrylamide gel electrophoresis (SDS- hibitor treatment was seeded into 6-well plates PAGE) for 2 h, and transferred onto 0.45 μm at a concentration of 300 cells per well. After 15 nitrocellulose membranes (Millipore, Billerica, days of culture in Dulbecco’s Modified Eagle’s MA, USA). After blocking with 5% skim milk, Medium (DMEM) containing 10% fetal bovine the membranes were incubated with prima- serum (FBS), formed colonies were fixed with ry antibodies of YWHAZ and glyceraldehyde methanol and stained with crystal violet. Final- 3-phosphate dehydrogenase (GAPDH) (Abcam, ly, the number of colonies with over 50 cells was Cambridge, MA, USA) overnight. On the next counted and recorded. day, the membranes were washed with phos- phate-buffered saline and Tween-20 (PBST) Transwell Assay solution (Beyotime, Shanghai, China), followed For invasion and migration assays, 8 μm by incubation with corresponding secondary pore size inserts (Millipore, Billerica, MA, antibody (Abcam, Cambridge, MA, USA) for USA) were used. For cell invasion, the cham- 1 h at room temperature. After the membrane ber was pre-coated with matrigel (BD, Frank- was completely washed, immunoreactive bands lin Lakes, NJ, USA). HepG2 or Huh-7 cells were exposed using enhanced chemilumines- transfected with miR-451a mimics or inhibitor cence (ECL) luminescent solution (Millipore, were seeded into the upper chamber at a densi- Billerica, MA, USA).