- Email: [email protected]
miR-204-5p regulates cell proliferation and metastasis through inhibiting CXCR4 expression in OSCC
Biomedicine & Pharmacotherapy 82 (2016) 202–207
Available online at
ScienceDirect www.sciencedirect.com
miR-204-5p regulates cell proliferation and metastasis through inhibiting CXCR4 expression in OSCC Xinjuan Wang* , Fujun Li, Xiaoli Zhou Department of Stomatology, The First Affiliated Hospital of Henan University of Science and Technology, Luoyang, China
A R T I C L E I N F O
Article history: Received 6 March 2016 Received in revised form 26 April 2016 Accepted 27 April 2016 Keywords: miR-204-5p OSCC CXCR4
A B S T R A C T
MicroRNAs (miRNAs) are important small molecules in cancer including oral squamous cell carcinoma (OSCC) which regulate gene expression at post-transcriptional levels. MiR-204-5p acts as a tumor suppressor in some of cancers, but the role of it in OSCC is not known. The aim of this study is to investigate the expression and functional roles of miR-204-5p in OSCC. The results showed that the expression of miR-204-5p was lower in cancer tissues or cells. Next, cell proliferation, cell cycle, migration and invasion were detected. It was found that miR-204-5p could enhance OSCC cell proliferation and metastasis. MiR-204-5p was predicted as a regulatory miRNA of CXCR4 in OSCC, and the data analysis indicated that there was a negatively relationship between miR-204-5p and CXCR4 expression in OSCC tissues from the patients. In a conclusion, our findings suggested that miR-204-5p may function as an inhibitory RNA molecule in OSCC by targeting CXCR4. ã 2016 Published by Elsevier Masson SAS.
1. Introduction It is reported that there are many patients die of oral squamous cell carcinoma (OSCC) every year in the world [1]. OSCC, like other cancers, is a multifactorial disease with complicated epigenetic background. The major risk factors of OSCC includes chronic tobacco, chronic inflammation, virus infection, betel quid chewing and genetic predisposition are supplementary factors that contribute towards its pathogenesis [2]. There is a low five-year survival rate in OSCC. In fact, the therapeutic methods for OSCC including surgery, radiotherapy or chemotherapy are not effective for patients with advanced OSCC due to tumor recurrence, metastasis, and poor response to chemotherapy and radiotherapy. To find new therapeutic methods, there needs to elucidate the further molecular mechanism of OSCC. Accumulating evidence indicates that microRNA (miRNAs) play an important regulatory role in the initiation and progression of OSCC [2]. MiRNAs are a class of approximately 22 nucleotide non-coding small RNAs, which can down-regulate target mRNAs by binding to their 30 -untranslated region (30 -UTR) [3]. MiRNAs regulate gene expression on a post-transcriptional level, inhibiting protein formation by degradation or repression of translation of the
mRNA transcript [4]. There are reports showing that miRNAs participate in multiple physiological and pathological processes of OSCC, such as miR-21, miR-31, miR-146a and miR-155 play as an oncogene [4]. However, like miR-7, miR-29a and miR-218 play as a suppressor in OSCC [4]. These miRNAs work as targets of the genes that involve in cell proliferation, metastasis and cancer related signal pathways, which modulate genomic instability, cellular metabolism, or angiogenesis, which in turn promote malignant progression. miR-204-5p may play a suppressing role in several type of cancer [5–12]. However, there is no report in OSCC. In the current study, it focused on the expression of miR-204-5p in OSCC and its role in cell proliferation, cell cycle and metastasis. Our results identified a group of aberrantly expressed miRNAs and confirmed that miR-204-5p is a suppressive miRNA involved in malignant transformation. In addition, we found that miR-204-5p can bind directly to the 30 -UTR of CXCR4, an important chemokine receptor widely expressed in tumor cell surface. The data demonstrate that CXCR4 is regulated by miR-205-5p in OSCC. 2. Materials and methods 2.1. Patients and tumor tissues
* Corresponding author at: 24 Jinghua Road, Department of Stomatology, The First Affiliated Hospital of Henan University of Science and Technology, Luoyang, Henan, 471003, China. E-mail address: [email protected] (X. Wang). http://dx.doi.org/10.1016/j.biopha.2016.04.060 0753-3322/ã 2016 Published by Elsevier Masson SAS.
A total of 52 frozen OSCC patient specimens were selected. These patients had undergone surgical resection of primary OSCC at the First Affiliated Hospital of Henan University of Science and
X. Wang et al. / Biomedicine & Pharmacotherapy 82 (2016) 202–207
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Fig. 1. The expression of miR-204-5p in OSCC tissues and cells. (A) miR-204-5p expression in OSCC tissues and the normal tissues was examined by qRT-PCR. (B) miR-204-5p expression in OSCC cell lines was examined by qRT-PCR. *p < 0.05, **p < 0.01 vs control.
Technology (Luoyang, China) under the procedures approved by the Ethnic Committee for Use of Human Samples of Henan University of Science and Technology. 2.2. Cell lines and culture Human OSCC cell lines were primarily obtained from American Type Culture Collection and were maintained in RPMI-1640 medium supplemented with fetal bovine serum (Biological Industries) and antibiotics at 37 C in a 5% CO2 incubator. 2.3. Real time RT-PCR Total RNA was isolated from OSCC tissues or cells using Trizol (Invitrogen, Carlsbad, CA, USA). Total RNA was subjected to
first-strand cDNA synthesis for 15 min at 37 C and 5 s at 85 C using a PrimeScript RT Reagent kit (TaKaRa, Japan). The qPCR was performed using SYBR Green PCR master mix (TaKaRa, Japan) on the ABI 7900HT System. The U6 or GAPDH were used as an endogenous control. The relative fold expression was calculated with the 2DDCT method. 2.4. Cell proliferation assay OSCC cells were transfected with miR-204-5p and then plated in 96-well plates. At 48 h after transfection, the cells were incubated with MTT (Sigma) for 4 h at 37 C. Then the cells were agitated with MTT solvent on an orbital shaker for 10 min avoiding light. The absorbance at 570 nm was measured using a Quant Universal Microplate Spectropho-tometer (BioTek, Winooski, VT).
Fig. 2. MiR-204-5p suppressed OSCC cellular growth in vitro. (A) miR-204-5p expression in OSCC cells transfected with miR-204-5p mimics. (B–C) Cell proliferation was assayed in OSCC cells with miR-204-5p transfection. (D) Cell cycle in OSCC cells transfected with miR-204-5p mimics by flow cytometry. (E–F) Data analysis from D. (G) Western blotting of Ki67 and cyclin D1 in OSCC cells transfected with miR-204-5p mimics. *p < 0.05, **p < 0.01 vs control.
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Fig. 3. MiR-204-5p inhibited the OSCC cell metastasis. (A) Cell migration was assayed in OSCC cells with miR-204-5p transfection. (B) Data analysis from A. (C) Cell invasion was assayed in OSCC cells with miR-204-5p transfection. (D) Data analysis from C. **p < 0.01 vs control.
2.5. Cell cycle analysis OC-3 and HSC-2 cells were transfected miR-204-5p for 24 h and digested for cell cycle analysis. Cells were washed using cold PBS and fixed in 70% ethanol overnight at 4 C. Fixed cells were washed with PBS and incubated with RNase A and PI for 30 min. The DNA content was determined using a FACSCanto flow cytometer (BD Biosciences, USA) and the cell cycle profile was evaluated by FACS Diva Software. 2.6. Wound healing assay For the wound healing assay, OC-3 and HSC-2 cells were seeded into a 6-well plate and transfected miR-204-5p for 24 h. When the cell confluence reached approximately 80% post-transfection, scratches were made by using a 100 ml pipette tip. After incubation for 48 h, detached cells were washed and 3 random fields were picked for take photos.
plated on the upper chamber at 37 C with the growth medium containing 0.1% FBS and the low well contained 10% FBS. After 24 h, the non-invading cells were removed with cotton swabs. The invaded cells at the bottom of the membrane were stained with 0.1% crystal violet and were counted under microscopy. 2.8. Western blot The protein was extracted from the OSCC specimen or adjacent normal tissue, or cultured cells. Protein extraction, SDS-PAGE gel electrophoresis and blotting were performed as we previously described. Several different primary antibodies were used including: CXCR4 (Cell Signaling Technology, Danvers, USA) and GAPDH (Santa Cruz Biotechnology, Santa Cruz, USA). The secondary antibody incubations were performed for 2 h at room temperature and protein bands were visualized using an enhanced ECL system. 2.9. Dual luciferase assay
2.7. Invasion assay Invasion assay was performed using a transwell chamber system (24-well plates, Corning). Transfected OSCC cells were
A total of 100 ng wild type (WT) or mutant (MUT) reporter and miR-204-5p were co-transfected into the OSCC cells with Lipofectamine 2000 transfection reagent based on the instruction.
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Fig. 4. CXCR4 is a direct target of miR-204-5p in OSCC cells. (A) Wild typed and mutant target sites for miR-204-5p sequences in 30 UTR of CXCR4 were shown. (B) Luciferase reporter assays were performed to verify the binding of miR-204-5p in 30 -UTR of CXCR4. W: wild type; M: mutation. (C) qRT-PCR assay was performed to detect the mRNA level of CXCR4 in OC-3 and HSC-2 cells treated with miR-204-5p mimics. MiR-204-5p was measured by real-time RT-PCR in OC-3 and HSC-2 cells after miR-204-5p mimic transfection. (D) Western blotting analysis was used to measure CXCR4 protein in OC-3 and HSC-2 cells treated with miR-204-5p mimics. **p < 0.01 vs control.
After 48 h, luciferase activity was measured with the DualLuciferase reporter assay system (Promega). 2.10. Statistical analysis The results are presented as means standard deviation (SD). Statistical significance was determined using Student’s t-test. P-value < 0.05 was considered statistically significant. 3. Results 3.1. Significance of miR-204-5p in OSCC patients and cells To elucidate the expression of miR-204-5p in human OSCC, miR-204-5p was detected in 52 pairs of OSCC tissues and their matched noncancerous oral tissues using a qRT-PCR method, as well as OSCC cell lines. Low expression of miR-204-5p was shown in cancer tissues as compared to that of the normal tissues (Fig. 1A). Meanwhile, the miR-204-5p expression was significantly
decreased in all five OSCC cell lines examined as compared to that of the normal cells (Fig. 1B). Taken together, these data provided an evidence that the role of miR-204-5p in OSCC is negatively related to the progression. 3.2. miR-204-5p suppressed OSCC cellular growth To investigate the role of miR-204-5p on OSCC cells, OSCC cells were transfected with miR-204-5p mimics. The expression of miR204-5p was enhanced in the cells with miR-204-5p mimics transfection (Fig. 2A). After transfection with miR-204-5p, the proliferation was significantly inhibited in OSCC cells (Fig. 2B and C). The analysis of cell cycle in OSCC cells showed that miR-204-5p could lead to significantly reduce the proportion of G0/G1 in both of the cell lines and G2/M phase in OC-3 cells. Furthermore, they increased the proportion of S phase in OSCC cells as compared to that of the controls (Fig. 2D–F). In addition, when the OSCC cells were over-expressed miR-204-5p, cell proliferation marker Ki67 was down-regulated, so did cell cycle protein cyclin D1
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Fig. 5. Relationship of miR-204-5p and CXCR4 in the clinical OSCC. (A) Data analysis of miR-204-5p expression in OSCC tissues with non-metastasis and metastasis OSCC tissues. (B) Data analysis of relationship between miR-204-5p and CXCR4 expression in OSCC tissues (r = 0.463). **p < 0.01.
(Fig. 2G). The above results indicated that miR-204-5p suppresses OSCC cell proliferation. 3.3. MiR-204-5p inhibited the OSCC cell metastasis The effect of miR-204-5p on the OSCC cell invasiveness was investigated by Matrigel invasion assays. We found that the number of migrated OSCC cells transfected with the miR-204-5p mimics was remarkably lower than the number of those transfected with miRNA control (Fig. 3A and B). Similar results were also obtained for the invasiveness of the cells transfected with the miR204-5p mimics (Fig. 3C and D). It revealed that the miR-204-5p mediated the inhibition of OSCC metastasis. 3.4. CXCR4 is regulated by miR-204-5p in OSCC cells The predicted result showed that CXCR4, a key oncogene, as one of the potential targets for miR-204-5p (Fig. 4A). To verify the prediction, the luciferase report plasmids with the target sequences of CXCR4 30 UTR (wild type, WT) or the mutant sequence (mutant type, MUT) were constructed. When the OC-3 cells were co-transfected with the luciferase vectors combined with miR204-5p, it was found that the firefly luciferase activity in the reporter with wild type 30 UTR was significantly inhibited, however, but the activity of mutant 30 UTR vector had no change (Fig. 4B). It was also found that CXCR4 mRNA expression levels OSCC cells reduced using qRT-PCR (Fig. 4C). The result from western blotting showed that CXCR4 protein levels decreased (Fig. 4D). 3.5. MiR-204-5p was negatively correlated to CXCR4 in OSCC tissues To further investigate the relationship miR-204-5p to OSCC clinical features, we found in the non-metastasis OSCC tissues, miR-204-5p expression was relatively higher than in the metastasis OSCC tissues (Fig. 5A). Further analysis showed that a significant inverse correlation was observed between CXCR4 mRNA and miR-204-5p expression levels in OSCC tissues (Fig. 5B). 4. Discussion As we all know, miRNAs play important roles in cancer progression, however, its expression profile in cancer genome is largely unknown. The function or expression of specific miRNA is OSCC is still unclear. Here, we focused miR-204-5p, which is not studied in OSCC. The functional role or miR-204-5p in tumor was studied in breast cancer [5], gastric cancer [6,7], glioma [8], papillary thyroid carcinoma [9], colorectal cancer [10,11] and endometrial carcinoma [12]. miR-204-5p expression levels was down-regulated. The
results showed that it can inhibit EMT of breast cancer cells by targeting Six1 [5], cell proliferation of gastric cancer by targeting USP47 and RAB22A [7,8], cell proliferation of papillary thyroid carcinoma by targeting IGFBP5 [5], cell proliferation, metastasis of colorectal cancer by targeting RAB22 [10]. In addition, miR-204-5p could enhance the chemotherapy sensitivity in colorectal cancer [10]. Our results showed that the expression levels of miR-204-5p decreased in most of OSCC tissues and cancer cell lines. However, in the other OSCC tissues and cell lines, miR-204-5p levels changed not much or increased. In the two OSCC cell lines used in our study, introduction of miR-204-5p into the cells could lead to the inhibition of OSCC cell proliferation and metastasis. The chemokine receptor CXCR4 and its ligand CXCL12 plays important roles in the initiation or progression of cancers. In OSCC, there are limited reports showed that overexpressed CXCR4 was found, which was related to its malignant progression including cell cycle arrest, apoptosis, proliferation, lymph node metastasis and metastasis [13–21]. CXCR4 promotes OSCC cell metastasis via Wnt/b-catenin or NF-kappaB signaling pathways [18,19]. There are many miRNAs regulating CXCR4 expression in cancer such as miR146 [20], miR-222 [20], miR-302a [21], miR-133b [22], miR-9 [23,24] and let-7a [25]. In this study, we found miR-204-5p was a new regulator of CXCR4 gene expression in OSCC cells. Restoration of miR-204-5p could induce down-regulation of CXCR4 mRNA and protein in OSCC cells. CXCR4 is evaluated as a direct target gene of miR-204-5p. Altogether, our results indicate that miR-204-5p expression in OSCC tissues and cells is lower than normal controls, which regulates the survival of OSCC cells. CXCR4 is the target gene of miR-204-5p in OSCC. The study suggests that miR-204-5p may be a useful tool in OSCC therapeutics.
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X. Wang et al. / Biomedicine & Pharmacotherapy 82 (2016) 202–207 [7] B. Zhang, Y. Yin, Y. Hu, J. Zhang, Z. Bian, M. Song, D. Hua, Z. Huang, MicroRNA204-5p inhibits gastric cancer cell proliferation by downregulating USP47 and RAB22A, Med. Oncol. 32 (1) (2015) 331. [8] Z. Xia, F. Liu, J. Zhang, L. Liu, Decreased expression of miRNA-204-5p contributes to glioma progression and promotes glioma cell growth, migration and invasion, PLoS One 10 (7) (2015) e0132399. [9] L. Liu, J. Wang, X. Li, J. Ma, C. Shi, H. Zhu, Q. Xi, J. Zhang, X. Zhao, M. Gu, MiR-2045p suppresses cell proliferation by inhibiting IGFBP5 in papillary thyroid carcinoma, Biochem. Biophys. Res. Commun. 457 (4) (2015) 621–626. [10] Y. Yin, B. Zhang, W. Wang, B. Fei, C. Quan, J. Zhang, M. Song, Z. Bian, Q. Wang, S. Ni, Y. Hu, Y. Mao, L. Zhou, Y. Wang, J. Yu, X. Du, D. Hua, Z. Huang, miR-204-5p inhibits proliferation and invasion and enhances chemotherapeutic sensitivity of colorectal cancer cells by downregulating RAB22A, Clin. Cancer Res. 20 (23) (2014) 6187–6199. ebakan, S. Ergün, E. Yengil, C.Y. Batmacı, Ö. Tonyalı, M. [11] A.T. Sümbül, B. Gög Yaldız, miR-204-5p expression in colorectal cancer: an autophagy-associated gene, Tumour Biol. 35 (12) (2014) 12713–12719. [12] W. Bao, H.H. Wang, F.J. Tian, X.Y. He, M.T. Qiu, J.Y. Wang, H.J. Zhang, L.H. Wang, X.P. Wan, A TrkB-STAT3-miR-204-5p regulatory circuitry controls proliferation and invasion of endometrial carcinoma cells, Mol. Cancer 12 (2013) 155. [13] N. Chen, X. Jiang, J. Wang, T. Wu, B. Cheng, J. Xia, CXCL12-CXCR4/CXCR7 axis contributes to cell motilities of oral squamous cell carcinoma, Tumour Biol. (August) (2015). [14] J.I. Lee, B.H. Jin, M.A. Kim, H.J. Yoon, S.P. Hong, S.D. Hong, Prognostic significance of CXCR-4 expression in oral squamous cell carcinoma, Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 107 (5) (2009) 678–684. [15] J. Xia, N. Chen, Y. Hong, X. Chen, X. Tao, B. Cheng, Y. Huang, Expressions of CXCL12/CXCR4 in oral premalignant and malignant lesions, Mediators Inflamm. 2012 (2012) 516395. [16] T. Yu, Y. Wu, Y. Huang, C. Yan, Y. Liu, Z. Wang, X. Wang, Y. Wen, C. Wang, L. Li, RNAi targeting CXCR4 inhibits tumor growth through inducing cell cycle arrest and apoptosis, Mol. Ther. 20 (2) (2012) 398–407.
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Available online at
ScienceDirect www.sciencedirect.com
miR-204-5p regulates cell proliferation and metastasis through inhibiting CXCR4 expression in OSCC Xinjuan Wang* , Fujun Li, Xiaoli Zhou Department of Stomatology, The First Affiliated Hospital of Henan University of Science and Technology, Luoyang, China
A R T I C L E I N F O
Article history: Received 6 March 2016 Received in revised form 26 April 2016 Accepted 27 April 2016 Keywords: miR-204-5p OSCC CXCR4
A B S T R A C T
MicroRNAs (miRNAs) are important small molecules in cancer including oral squamous cell carcinoma (OSCC) which regulate gene expression at post-transcriptional levels. MiR-204-5p acts as a tumor suppressor in some of cancers, but the role of it in OSCC is not known. The aim of this study is to investigate the expression and functional roles of miR-204-5p in OSCC. The results showed that the expression of miR-204-5p was lower in cancer tissues or cells. Next, cell proliferation, cell cycle, migration and invasion were detected. It was found that miR-204-5p could enhance OSCC cell proliferation and metastasis. MiR-204-5p was predicted as a regulatory miRNA of CXCR4 in OSCC, and the data analysis indicated that there was a negatively relationship between miR-204-5p and CXCR4 expression in OSCC tissues from the patients. In a conclusion, our findings suggested that miR-204-5p may function as an inhibitory RNA molecule in OSCC by targeting CXCR4. ã 2016 Published by Elsevier Masson SAS.
1. Introduction It is reported that there are many patients die of oral squamous cell carcinoma (OSCC) every year in the world [1]. OSCC, like other cancers, is a multifactorial disease with complicated epigenetic background. The major risk factors of OSCC includes chronic tobacco, chronic inflammation, virus infection, betel quid chewing and genetic predisposition are supplementary factors that contribute towards its pathogenesis [2]. There is a low five-year survival rate in OSCC. In fact, the therapeutic methods for OSCC including surgery, radiotherapy or chemotherapy are not effective for patients with advanced OSCC due to tumor recurrence, metastasis, and poor response to chemotherapy and radiotherapy. To find new therapeutic methods, there needs to elucidate the further molecular mechanism of OSCC. Accumulating evidence indicates that microRNA (miRNAs) play an important regulatory role in the initiation and progression of OSCC [2]. MiRNAs are a class of approximately 22 nucleotide non-coding small RNAs, which can down-regulate target mRNAs by binding to their 30 -untranslated region (30 -UTR) [3]. MiRNAs regulate gene expression on a post-transcriptional level, inhibiting protein formation by degradation or repression of translation of the
mRNA transcript [4]. There are reports showing that miRNAs participate in multiple physiological and pathological processes of OSCC, such as miR-21, miR-31, miR-146a and miR-155 play as an oncogene [4]. However, like miR-7, miR-29a and miR-218 play as a suppressor in OSCC [4]. These miRNAs work as targets of the genes that involve in cell proliferation, metastasis and cancer related signal pathways, which modulate genomic instability, cellular metabolism, or angiogenesis, which in turn promote malignant progression. miR-204-5p may play a suppressing role in several type of cancer [5–12]. However, there is no report in OSCC. In the current study, it focused on the expression of miR-204-5p in OSCC and its role in cell proliferation, cell cycle and metastasis. Our results identified a group of aberrantly expressed miRNAs and confirmed that miR-204-5p is a suppressive miRNA involved in malignant transformation. In addition, we found that miR-204-5p can bind directly to the 30 -UTR of CXCR4, an important chemokine receptor widely expressed in tumor cell surface. The data demonstrate that CXCR4 is regulated by miR-205-5p in OSCC. 2. Materials and methods 2.1. Patients and tumor tissues
* Corresponding author at: 24 Jinghua Road, Department of Stomatology, The First Affiliated Hospital of Henan University of Science and Technology, Luoyang, Henan, 471003, China. E-mail address: [email protected] (X. Wang). http://dx.doi.org/10.1016/j.biopha.2016.04.060 0753-3322/ã 2016 Published by Elsevier Masson SAS.
A total of 52 frozen OSCC patient specimens were selected. These patients had undergone surgical resection of primary OSCC at the First Affiliated Hospital of Henan University of Science and
X. Wang et al. / Biomedicine & Pharmacotherapy 82 (2016) 202–207
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Fig. 1. The expression of miR-204-5p in OSCC tissues and cells. (A) miR-204-5p expression in OSCC tissues and the normal tissues was examined by qRT-PCR. (B) miR-204-5p expression in OSCC cell lines was examined by qRT-PCR. *p < 0.05, **p < 0.01 vs control.
Technology (Luoyang, China) under the procedures approved by the Ethnic Committee for Use of Human Samples of Henan University of Science and Technology. 2.2. Cell lines and culture Human OSCC cell lines were primarily obtained from American Type Culture Collection and were maintained in RPMI-1640 medium supplemented with fetal bovine serum (Biological Industries) and antibiotics at 37 C in a 5% CO2 incubator. 2.3. Real time RT-PCR Total RNA was isolated from OSCC tissues or cells using Trizol (Invitrogen, Carlsbad, CA, USA). Total RNA was subjected to
first-strand cDNA synthesis for 15 min at 37 C and 5 s at 85 C using a PrimeScript RT Reagent kit (TaKaRa, Japan). The qPCR was performed using SYBR Green PCR master mix (TaKaRa, Japan) on the ABI 7900HT System. The U6 or GAPDH were used as an endogenous control. The relative fold expression was calculated with the 2DDCT method. 2.4. Cell proliferation assay OSCC cells were transfected with miR-204-5p and then plated in 96-well plates. At 48 h after transfection, the cells were incubated with MTT (Sigma) for 4 h at 37 C. Then the cells were agitated with MTT solvent on an orbital shaker for 10 min avoiding light. The absorbance at 570 nm was measured using a Quant Universal Microplate Spectropho-tometer (BioTek, Winooski, VT).
Fig. 2. MiR-204-5p suppressed OSCC cellular growth in vitro. (A) miR-204-5p expression in OSCC cells transfected with miR-204-5p mimics. (B–C) Cell proliferation was assayed in OSCC cells with miR-204-5p transfection. (D) Cell cycle in OSCC cells transfected with miR-204-5p mimics by flow cytometry. (E–F) Data analysis from D. (G) Western blotting of Ki67 and cyclin D1 in OSCC cells transfected with miR-204-5p mimics. *p < 0.05, **p < 0.01 vs control.
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Fig. 3. MiR-204-5p inhibited the OSCC cell metastasis. (A) Cell migration was assayed in OSCC cells with miR-204-5p transfection. (B) Data analysis from A. (C) Cell invasion was assayed in OSCC cells with miR-204-5p transfection. (D) Data analysis from C. **p < 0.01 vs control.
2.5. Cell cycle analysis OC-3 and HSC-2 cells were transfected miR-204-5p for 24 h and digested for cell cycle analysis. Cells were washed using cold PBS and fixed in 70% ethanol overnight at 4 C. Fixed cells were washed with PBS and incubated with RNase A and PI for 30 min. The DNA content was determined using a FACSCanto flow cytometer (BD Biosciences, USA) and the cell cycle profile was evaluated by FACS Diva Software. 2.6. Wound healing assay For the wound healing assay, OC-3 and HSC-2 cells were seeded into a 6-well plate and transfected miR-204-5p for 24 h. When the cell confluence reached approximately 80% post-transfection, scratches were made by using a 100 ml pipette tip. After incubation for 48 h, detached cells were washed and 3 random fields were picked for take photos.
plated on the upper chamber at 37 C with the growth medium containing 0.1% FBS and the low well contained 10% FBS. After 24 h, the non-invading cells were removed with cotton swabs. The invaded cells at the bottom of the membrane were stained with 0.1% crystal violet and were counted under microscopy. 2.8. Western blot The protein was extracted from the OSCC specimen or adjacent normal tissue, or cultured cells. Protein extraction, SDS-PAGE gel electrophoresis and blotting were performed as we previously described. Several different primary antibodies were used including: CXCR4 (Cell Signaling Technology, Danvers, USA) and GAPDH (Santa Cruz Biotechnology, Santa Cruz, USA). The secondary antibody incubations were performed for 2 h at room temperature and protein bands were visualized using an enhanced ECL system. 2.9. Dual luciferase assay
2.7. Invasion assay Invasion assay was performed using a transwell chamber system (24-well plates, Corning). Transfected OSCC cells were
A total of 100 ng wild type (WT) or mutant (MUT) reporter and miR-204-5p were co-transfected into the OSCC cells with Lipofectamine 2000 transfection reagent based on the instruction.
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Fig. 4. CXCR4 is a direct target of miR-204-5p in OSCC cells. (A) Wild typed and mutant target sites for miR-204-5p sequences in 30 UTR of CXCR4 were shown. (B) Luciferase reporter assays were performed to verify the binding of miR-204-5p in 30 -UTR of CXCR4. W: wild type; M: mutation. (C) qRT-PCR assay was performed to detect the mRNA level of CXCR4 in OC-3 and HSC-2 cells treated with miR-204-5p mimics. MiR-204-5p was measured by real-time RT-PCR in OC-3 and HSC-2 cells after miR-204-5p mimic transfection. (D) Western blotting analysis was used to measure CXCR4 protein in OC-3 and HSC-2 cells treated with miR-204-5p mimics. **p < 0.01 vs control.
After 48 h, luciferase activity was measured with the DualLuciferase reporter assay system (Promega). 2.10. Statistical analysis The results are presented as means standard deviation (SD). Statistical significance was determined using Student’s t-test. P-value < 0.05 was considered statistically significant. 3. Results 3.1. Significance of miR-204-5p in OSCC patients and cells To elucidate the expression of miR-204-5p in human OSCC, miR-204-5p was detected in 52 pairs of OSCC tissues and their matched noncancerous oral tissues using a qRT-PCR method, as well as OSCC cell lines. Low expression of miR-204-5p was shown in cancer tissues as compared to that of the normal tissues (Fig. 1A). Meanwhile, the miR-204-5p expression was significantly
decreased in all five OSCC cell lines examined as compared to that of the normal cells (Fig. 1B). Taken together, these data provided an evidence that the role of miR-204-5p in OSCC is negatively related to the progression. 3.2. miR-204-5p suppressed OSCC cellular growth To investigate the role of miR-204-5p on OSCC cells, OSCC cells were transfected with miR-204-5p mimics. The expression of miR204-5p was enhanced in the cells with miR-204-5p mimics transfection (Fig. 2A). After transfection with miR-204-5p, the proliferation was significantly inhibited in OSCC cells (Fig. 2B and C). The analysis of cell cycle in OSCC cells showed that miR-204-5p could lead to significantly reduce the proportion of G0/G1 in both of the cell lines and G2/M phase in OC-3 cells. Furthermore, they increased the proportion of S phase in OSCC cells as compared to that of the controls (Fig. 2D–F). In addition, when the OSCC cells were over-expressed miR-204-5p, cell proliferation marker Ki67 was down-regulated, so did cell cycle protein cyclin D1
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Fig. 5. Relationship of miR-204-5p and CXCR4 in the clinical OSCC. (A) Data analysis of miR-204-5p expression in OSCC tissues with non-metastasis and metastasis OSCC tissues. (B) Data analysis of relationship between miR-204-5p and CXCR4 expression in OSCC tissues (r = 0.463). **p < 0.01.
(Fig. 2G). The above results indicated that miR-204-5p suppresses OSCC cell proliferation. 3.3. MiR-204-5p inhibited the OSCC cell metastasis The effect of miR-204-5p on the OSCC cell invasiveness was investigated by Matrigel invasion assays. We found that the number of migrated OSCC cells transfected with the miR-204-5p mimics was remarkably lower than the number of those transfected with miRNA control (Fig. 3A and B). Similar results were also obtained for the invasiveness of the cells transfected with the miR204-5p mimics (Fig. 3C and D). It revealed that the miR-204-5p mediated the inhibition of OSCC metastasis. 3.4. CXCR4 is regulated by miR-204-5p in OSCC cells The predicted result showed that CXCR4, a key oncogene, as one of the potential targets for miR-204-5p (Fig. 4A). To verify the prediction, the luciferase report plasmids with the target sequences of CXCR4 30 UTR (wild type, WT) or the mutant sequence (mutant type, MUT) were constructed. When the OC-3 cells were co-transfected with the luciferase vectors combined with miR204-5p, it was found that the firefly luciferase activity in the reporter with wild type 30 UTR was significantly inhibited, however, but the activity of mutant 30 UTR vector had no change (Fig. 4B). It was also found that CXCR4 mRNA expression levels OSCC cells reduced using qRT-PCR (Fig. 4C). The result from western blotting showed that CXCR4 protein levels decreased (Fig. 4D). 3.5. MiR-204-5p was negatively correlated to CXCR4 in OSCC tissues To further investigate the relationship miR-204-5p to OSCC clinical features, we found in the non-metastasis OSCC tissues, miR-204-5p expression was relatively higher than in the metastasis OSCC tissues (Fig. 5A). Further analysis showed that a significant inverse correlation was observed between CXCR4 mRNA and miR-204-5p expression levels in OSCC tissues (Fig. 5B). 4. Discussion As we all know, miRNAs play important roles in cancer progression, however, its expression profile in cancer genome is largely unknown. The function or expression of specific miRNA is OSCC is still unclear. Here, we focused miR-204-5p, which is not studied in OSCC. The functional role or miR-204-5p in tumor was studied in breast cancer [5], gastric cancer [6,7], glioma [8], papillary thyroid carcinoma [9], colorectal cancer [10,11] and endometrial carcinoma [12]. miR-204-5p expression levels was down-regulated. The
results showed that it can inhibit EMT of breast cancer cells by targeting Six1 [5], cell proliferation of gastric cancer by targeting USP47 and RAB22A [7,8], cell proliferation of papillary thyroid carcinoma by targeting IGFBP5 [5], cell proliferation, metastasis of colorectal cancer by targeting RAB22 [10]. In addition, miR-204-5p could enhance the chemotherapy sensitivity in colorectal cancer [10]. Our results showed that the expression levels of miR-204-5p decreased in most of OSCC tissues and cancer cell lines. However, in the other OSCC tissues and cell lines, miR-204-5p levels changed not much or increased. In the two OSCC cell lines used in our study, introduction of miR-204-5p into the cells could lead to the inhibition of OSCC cell proliferation and metastasis. The chemokine receptor CXCR4 and its ligand CXCL12 plays important roles in the initiation or progression of cancers. In OSCC, there are limited reports showed that overexpressed CXCR4 was found, which was related to its malignant progression including cell cycle arrest, apoptosis, proliferation, lymph node metastasis and metastasis [13–21]. CXCR4 promotes OSCC cell metastasis via Wnt/b-catenin or NF-kappaB signaling pathways [18,19]. There are many miRNAs regulating CXCR4 expression in cancer such as miR146 [20], miR-222 [20], miR-302a [21], miR-133b [22], miR-9 [23,24] and let-7a [25]. In this study, we found miR-204-5p was a new regulator of CXCR4 gene expression in OSCC cells. Restoration of miR-204-5p could induce down-regulation of CXCR4 mRNA and protein in OSCC cells. CXCR4 is evaluated as a direct target gene of miR-204-5p. Altogether, our results indicate that miR-204-5p expression in OSCC tissues and cells is lower than normal controls, which regulates the survival of OSCC cells. CXCR4 is the target gene of miR-204-5p in OSCC. The study suggests that miR-204-5p may be a useful tool in OSCC therapeutics.
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