microRNA-30b/c inhibits non-small cell lung cancer cell proliferation by targeting Rab18
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MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptional regulate gene expression in a variety of cancers. Increasing evidences indicate that miR-30 expression is down-regulated in numerous human cancers including non-small cell lung cancer (NSCLC) which hypothesizes that miR-30 may play an important role in tumorigenesis. The aim of this study was to investigate the target gene of miR-30 and its roles in tumor growth of NSCLC.
Luciferase reporter assays were employed to validate regulation of a putative target of miR-30. The effect of miR-30 on endogenous levels of this target were subsequently confirmed via Western blot (WB). Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to determine the expression level of miR-30 in NSCLC specimens and adjacent non-tumor tissues. MTT assays were conducted to explore the impact of miR-30 overexpression on the proliferation of human NSCLC cells.
Both miR-30b and miR-30c (miR-30b/c) were found having target site in same region of Rab18 mRNA. Luciferase assays using a reporter carrying a putative miR-30b/c target site in the coding DNA sequence (CDS) region of Rab18 revealed that miR-30b/c directly targeted Rab18. Overexpression of miR-30b/c led to down-regulation of Rab18 in A549 and H23 cells at protein levels but not mRNA levels. Down-regulation of miR-30b/c and up-regulation of Rab18 protein levels were detected in NSCLC specimens compared with adjacent non-tumor tissues. Overexpression of miR-30b/c suppressed NSCLC cells growth. Knockdown of Rab18 by siRNA significantly inhibited the proliferation of NSCLC cells.
We demonstrated that miR-30b/c was down-regulated in NSCLC specimens compared with adjacent non-tumor tissues. miR-30b/c directly targeted and down-regulated Rab18 expression and inhibited NSCLC cells proliferation. These data indicated that miR-30b/c could serve as a tumor suppressor gene involved in NSCLC pathogenesis.
KeywordsmiR-30b miR-30c Proliferation Rab18 NSCLC
Non-small cell lung cancer
Quantitative reverse transcription-polymerase chain reaction
Fetal bovine serum.
Lung cancer is the most common cause of cancer-associated deaths worldwide, especially for male . NSCLC accounts for nearly 80% of lung cancers . NSCLC population has grown quickly over the past five years in China . Although surveillance and clinical treatment strategies have been improved, the 5-year survival of NSCLC patients after curative resection is reported to be only 30–60% . Therefore, elucidating the potential mechanism that mediate the initiation and progression of NSCLC is urgent and of great interest.
miRNAs are a class of small non-coding RNAs which plays an important role in post-transcriptional regulation in various biological processes. Mechanistically, miRNAs bind to their target mRNAs and cause translation to be blocked or mRNA degradation [5, 6]. Accumulating evidences have suggested that miRNAs play diverse roles in tumorigenesis and cancer progression [7, 8, 9, 10]. In recent years, miRNAs have received great attention in NSCLC research. Several deregulated miRNAs in NSCLCs such as miR-221, miR-222, miR-449a, miR-21, miR-205, miR-10b, miR-143 and miR-181a have been shown to regulate cell growth, apoptosis, migration and invasion [11, 12, 13, 14, 15, 16]. These findings indicate that deregulation of miRNA expression may be associated with tumorigenesis of NSCLCs.
miR-30 is significantly down-regulated in several cancers, including breast cancer , malignant peripheral nerve sheath tumors , glioma , and lung cancer . As the down-regulation of miR-30 is related to a number of cancers, it has been hypothesized that miR-30 may play an important role in tumorigenesis and tumor development. However, the function of miR-30 especially in NSCLC remains unclear.
In our study, we showed that Rab18 were identified as direct functional targets of miR-30b/c in NSCLC cells and miR-30b/c was down-regulated in NSCLC tissues compared to adjacent non-tumor tissues. Furthermore, ectopic overexpression of miR-30b/c blocked tumor cell proliferation in vitro. These data suggested that the reduced expression of miR-30b/c might facilitate the development of NSCLCs.
In this study, 5 paired NSCLC and adjacent non-tumor specimens were collected from the Department of Respiratory Medicine, the Second Affiliated Hospital, Second Military Medical University (Shanghai, China). All tissue samples were flash-frozen in liquid nitrogen immediately after collection and stored at -80°C until use. The study protocol was approved by Shanghai Changzheng Hospital Ethical Committee. Informed consent was obtained from all patients. All clinic pathologic and biological data were available for those patients. Both tumor and non-tumor samples were confirmed by pathological examination. No patients received chemotherapy or radiotherapy prior to surgery.
The human NSCLC cell lines A549 and H23 were purchased from ATCC. A549 cells were cultured in Ham’s F12K media (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (vol/vol) fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA), H23 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich, St. Louis, Mo., USA) supplemented with 10% (vol/vol) FBS, HEK293 cells were purchased from ATCC and grown were cultured in DMEM media containing 10% (vol/vol) FBS. Cells were maintained at 37°C in a humidified atmosphere with 5% CO2.
RNA isolation and qRT-PCR
All primers used in this study
Rab18 (WT) F
Rab18 (WT) R
Rab18 (MUT) F
Rab18 (MUT) R
miRNA Universal R
The Western blot protocol was described previously . Proteins were separated on a 12% SDS-PAGE gel and transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, USA). The membrane was blocked with 5% non-fat milk and incubated with anti-Rab18 antibody (Proteintech Group) or anti-beta-actin antibody (Sigma, CA, USA). After being washed extensively, secondary antibody (Pierce, IL, USA) was added to the system. Immunoreactive protein bands were detected using an Odyssey Scanning system.
RNA oligonucleotides were chemically synthesized and purified by Genepharma Co. Ltd., (Shanghai, China). Sequence of human miR-30b mimics was 5′- UGU AAA CAUC CUA CAC UCA GCU -3′ and human miR-30c mimics was 5′- UGU AAA CAU CCU ACA CUC UCA GC -3′. Negative control oligonucleotides for miRNA mimics was 5′-CAG UAC UUU UGU GUA GUA CAA-3′. The sequences of Rab18 siRNA was: 5′- GAA ACA UAC UGU ACA AGA ATT -3′ (sense) and 5′-UUC UUG UAC AGU AUG UUU CTT-3′ (antisense), Control siRNA was: 5′-UUC UCC GAA CGU GUC ACG UTT-3′ (sense), 5′-ACG UGA CAC GUU CGU AGA ATT-3′ (antisense). The transfections were performed with INTERFERin reagent (Polyplus-transfection). The final concentration of miRNA was 50 nM. The final concentration of siRNA was 20 nM.
Luciferase reporter construct was made by cloning human Rab18 sequence containing the potential miR-30b/c binding site into pMIR-Report construct (Ambion, Austin, USA). Wild type (WT) or mutant (MUT) Rab18 fragment (from 501 to 562) was amplified by PCR using the primers in Table 1. All fragments were amplified and cloned into the luciferase repoter via SpeI and HindIII sites. Luciferase reporter assays were performed as previously , HEK293 cells were co-transfected with 50 nM miRNA mimics or negative control oligonucleotides, 100 ng of firefly luciferase reporter and 20 ng of pRL-TK (Promega, USA) using the JetPRIME reagent (Polyplus-transfection). Cells were collected 48 hours after last transfection and analyzed using Dual-Luciferase Reporter Assay System (Promega).
The in vitro growth of NSCLC cells was measured using the MTT assay. 5000 cells were seeded into each well of 96-well plates and transfected with miRNA mimics or negative control oligonucleotides at a final concentration of 50 nM respectively. On the day of harvest, 100 μl of spent medium was replaced with an equal volume of fresh medium containing MTT 0.5 mg/ml. Plates were incubated at 37°C for 4 hrs, then the medium was replaced by 100 μl of DMSO (Sigma) and plates shaken at room temperature for 10 min. The absorbance was measured at 570 nm.
Immunohistochemical (IHC) staining of samples was performed as previously reported . Paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated in graded series of ethanol followed by heat induced epitope retrieval in citrate buffer (PH 6.0). Rab18 expression were detected using anti-Rab18 polyclonal antibody (Proteintech Group).
Data are presented as the mean ± standard deviation from at least three independent experiments. The two-tailed t-test was used to draw a comparison between groups. The null hypothesis was rejected at the 0.05 level.
miR-30b/c directly targets Rab18 in human NSCLC cells
miR-30b/c are low-expressed and Rab18 is high-expressed in NSCLC tissue samples
miR-30b/c inhibits NSCLC cell proliferation
miR-30b/c inhibits the proliferation of NSCLC cells via regulation of Rab18
Recently, attentions have focused on the role of miRNA in tumorigenesis. In this study, we focused on miR-30 which was decreased in several tumor types including NSCLC. Our results showed that Rab18 was one direct functional targets of miR-30b/c in NSCLC cells. Down-regulation of miR-30b/c and up-regulation of Rab18 protein levels were also found in NSCLC tissues compared to adjacent non-tumor tissues. Furthermore, ectopic overexpression of miR-30b/c blocked NSCLC cells proliferation in vitro.
The aberrant expression of miRNAs is associated with cancer progression including proliferation, migration, invasion and apoptosis. Deregulation of miRNAs such as miR-221, miR-222, miR-449a, miR-21, miR-205, miR-10b, miR-143 and miR-181a in NSCLC is a key factor underlying tumorigenesis . Human miR-30 is down-regulated in several tumor types including NSCLC . This suggests miR-30 is a potential tumor suppressor. These findings prompted us to investigate the regulation of miR-30 in NSCLC cells. Recent studies showed that miR-30a regulated growth of breast cancer cells , down-regulation of miR-30 maintained self-renewal and inhibited apoptosis in breast tumor-initiating cells , miR-30 regulated B-Myb expression during cellular senescence . However, the role of miR-30 in cancers especially in NSCLC is not very much known. In this study, we confirmed that oncogene Rab18 was directly targeted by miR-30b/c in NSCLC cells. Decreased miR-30b/c and increased Rab18 protein expression were also found in NSCLC tissues, which suggested that Rab18 was regulated by miR-30b/c in human NSCLC tissues. Human miR-30 family including miR-30a, miR-30b, miR-30c, miR-30d and miR-30e have the samilar sequence. Whether other miR-30 family have the samilar function like miR-30b and miR-30c in NSCLC or other cancer cells remain to be investigated.
Rabs, small G proteins belonging to the Ras superfamily, are regulators of vesicular transport in both exocytic and endocytic pathways in eukaryotic cells . Emerging evidences have revealed the association between dysfunction of the Rab18 and multiple human diseases including cancer [29, 30, 31]. It had been reported that Rab18 was involved in the lipogenesis of 3T3-L1 adipocytes . Loss-of-function mutations in Rab18 caused Warburg Micro syndrome . Moreover, Rab18 acted as a novel tumor antigen in medulloblastoma and HCC [23, 33]. However, the expression of Rab18 is less well known in human cancers especially in NSCLC. In this study, we first reported that Rab18 protein levels were highly expressed in NSCLC tissues compared to matched adjacent non-tumor tissues. To investigate the underlying mechanism of up-regulation of Rab18 protein levels in NSCLC. miRNA-binding sites analysis revealed that Rab18 was one direct functional target of miR-30b/c in NSCLC cells. Transfection of miR-30b/c mimics into NSCLC cells led to a significant Rab18 decrease at protein levels but not mRNA levels and inhibition of cellular proliferation. Furthermore, silencing Rab18 expression by siRNA in NSCLC cells also led to inhibition of cellular proliferation. These findings support the hypothesis that decreased expression of Rab18 by miR-30b/c accounts for the suppression of cellular proliferation in NSCLC. Our results agreed with the previous study in HCC which showed that Rab18 was directly targeted and down-regulated at protein levels by miR-429 in HCC cells, but whether the expression of Rab18 was also regulated at transcriptional levels in NSCLC cells just like that by hepatitis B virus X protein stimulation in HCC cells remain to be investigated .
Taken together, we demonstrate that miR-30b/c is down-regulated in NSCLC tissues. Overexpression of miR-30b/c directly down-regulates Rab18 and inhibits NSCLC cell proliferation. These data indicate that miR-30b/c could serve as a tumor suppressor gene involved in NSCLC pathogenesis.
This research was made possible with financial support from National Natural Sciences Foundation of China, for the project 81201780.
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