Upregulation of miR-205 induces CHN1 expression, which is associated with the aggressive behaviour of cervical cancer cells and correlated with lymph node metastasis

Human cervical cancer tumours and adjacent non-tumour tissues were obtained from Guangxi Medical University (China). The clinicopathological characteristics of the samples are summarised in Table 1. A cervical cancer tissue microarray was purchased from Shanghai Outdo Biotech Co. Ltd. (China). All patients provided informed consent for the use of their tissues before surgery. The study was approved by the Ethics Committee of the National Research Institute for Family Planning.

The human cervical carcinoma cell lines HeLa, SiHa, and C33A were purchased from the Cell Resource Center of Peking Union Medical College (Beijing, China) and cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% foetal bovine serum (FBS), 100 IU/mL penicillin, and 10 mg/mL streptomycin. All cells were maintained at 37 °C in an atmosphere containing 5% CO₂.

Sections (4 μm) of cervical cancer tissues and adjacent normal cervical tissues were dewaxed and rehydrated, followed by an antigen retrieval procedure (citrate buffer, pH 6.0; 95 °C heat for 15 min). For CHN1 staining, the sections were soaked in 3% H₂O₂ for 15 min and incubated overnight at 4 °C with rabbit anti-CHN1 antibodies (12048–1-AP; 1:150; Proteintech, USA). Matched rabbit nonimmune IgG was used as a negative control. The sections were then treated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (PV-6001; Zymed Laboratories, China) and incubated for 20 min at 37 °C; the proteins were visualised with 3,3′-diaminobenzidine tetrahydrochloride and counter stained with hematoxylin. Immunohistochemical analysis of CHN1 was carried out according to the “HSCORE” method [31]; an HSCORE of 75 or greater was considered positive. The specimens were analysed by two observers who were unaware of the patients’ clinical outcome. Discrepancies between the observers were found in < 10% of the slides examined, and consensus was reached on further review.

The miR-205 mimic, miR-205 inhibitor, corresponding negative control (NC), and siRNA duplex against human CHN1 were designed and synthesised by GenePharma (GenePharma Co., Ltd., Shanghai, China). Their sequences are shown in Table 2. Transient transfection was performed using lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

The 3′ untranslated regions (UTRs) of the human CHN1 gene (NM_001025201.2) were amplified by polymerase chain reaction (PCR) from human genomic DNA, cloned into the SbfI and NheI site of the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega), checked for orientation, and sequenced; the resulting plasmid was named pmirGLO-CHN1-wt. PCR primers used to amplify the CHN1 3’UTR are shown in Table 2. Site-directed mutagenesis of the miR-205 target site in the CHN1 3’UTR was carried out using an Easy Mutagenesis System (Transgen, China), with pmirGLO-CHN1-wt as a template; the resulting plasmid was named pmirGLO-CHN1-mut.

Next, 5 × 10⁴ cells were seeded in each well of a 48-well plate at 24 h before transfection. For reporter assays, the cells were transiently cotransfected with 0.25 μg wild-type (WT) or mutant reporter plasmid and 7.5 pmol NC or miR-205 mimic using Lipofectamine 2000. At 48 h after cotransfection, Firefly and Renilla luciferase activities were measured consecutively using Dual Luciferase Assays (Promega) according to the manufacturer’s instructions. Three independent experiments were performed.

Total RNA extraction and qRT-PCR experiments were performed for analysis of gene expression as follows. Briefly, RNA was isolated using TRIzol reagent (Invitrogen). For cDNA synthesis, approximately 2 μg of total RNA was used for reverse transcription with oligo-(dT)₁₈ primers using moloney murine leukaemia virus (M-MLV) reverse transcriptase (TaKaRa Bio, Otsu, Japan). The specific forward primer for miR-205 was designed by GenePharma based on the miRNA sequence from the miRbase database.

qRT-PCR was performed with an ABI Prism 7700 Sequence Detector System (PE Applied Biosystems, Foster City, CA, USA) using SYBR Premix Ex Taq II (TaKaRa Bio) and specific primers for each gene. To control for uniform amount of input RNA template, mRNA and miRNA expression results were normalised to the expression level of the internal control gene GAPDH or U6 snRNA, respectively. Thermal cycling conditions were as follows: an initial activation cycle at 95 °C for 30 s, followed by 40 cycles of denaturation (95 °C for 10 s), annealing, and amplification (60 °C for 30s). The final amplification products were verified by agarose gel electrophoresis for treatment samples and negative controls. The primer sequences are shown in Table 2. Each sample was assayed in triplicate. To compare the expression levels among different samples, relative quantification was achieved using the 2^-ΔΔCt approach, in which ΔΔCt is the calibrated Ct value.

Total protein lysates were obtained using RIPA lysis buffer supplemented with 1 mM PMSF, protease inhibitor cocktail, 1 mM Na₃VO₄, and 10 mM NaF (Sigma Aldrich, St. Louis, MO, USA). The protein concentrations in extracts were determined by colorimetric BCA protein assays (Thermo Scientific, USA). Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) on 10% Tris-glycine gels (Amresco, Solon, OH, USA) and then transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked for 1 h at RT with TBST (50 mM Tris-HCl, 150 mM NaCl, and 0.1% [v/v] Tween-20) containing 5% (w/v) nonfat dried milk and were subjected to immunoblotting with antibodies to CHN1 (12048–1-AP; Proteintech) and β-actin (CoWin, Beijing, China).

The proliferation of HeLa, SiHa, and C33A cells was estimated with a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Japan) according to the manufacturer’s instructions following transfection with miR-205 mimic, NC, miR-205 inhibitor, or inhibitor NC, with five wells for each treatment. The experiment was repeated three times, and the results are described as the ratio of the absorbance at 450 nm for the miR-205 mimic or inhibitor to that of the corresponding control.

Cell apoptosis was analysed using flow cytometry analysis with an Alexa Fluor 488 annexin V/Dead Cell Apoptosis Kit (Invitrogen). Samples containing 5 μL Alexa Fluor 488 annexin V and 1 μL of 100 μg/mL propidium iodide (PI) were assayed to determine the phosphatidylserine (PS) exposure on the outer leaflet of the plasma membrane. After incubation for 15 min at in a light-protected area, the specimens were quantified by flow cytometry (BD Biosciences, San Jose CA, USA). Each treatment was repeated twice, and the experiment was repeated three times.

HeLa, SiHa, and C33A cells were transfected with the miR-205 mimic, NC, miR-205 inhibitor, or inhibitor NC. Transfected cells were harvested and subjected to the following assays at 48 h after transfection. For migration assays, the transfected cells (0.5 × 10⁶ cells/mL) were seeded in the top of a chamber containing a membrane with 8.0-μm pores (Corning Costar Corp., Cambridge, MA, USA). Following a 12–18 h incubation period, cells that passed through the membrane were fixed and stained with hematoxylin. Cells were scraped and removed from the top of chamber. Membranes were mounted on cover slides, and cells were counted. Cell migration was quantified by counting the number of cells passing through the pores from five different randomly selected fields of view per sample at 100× magnification under a microscope. For invasion assays, Matrigel (BD Biosciences) diluted to 1 mg/mL in serum-free cold cell culture medium was added to the top of a chamber containing a membrane with 8.0-μm pores and incubated at 37 °C overnight until the matrigel solidified. Analysis was then carried out as described above. Samples were viewed under a Nikon TE 2000-U microscope (Nikon, Tokyo, Japan).

Expression patterns of miR-205 were analysed by in situ hybridisation in 46 pairs of human cervical cancer tissues and adjacent normal cervical tissues. As shown in Fig. 1, the expression of miR-205 was high in human cervical cancer tissues, but low or undetectable in normal cervical epithelium and noncancerous cervical stratified epithelium. Then, miR-205 target genes were searched using bioinformatic prediction of potential miR-205 binding sites with PicTar, Targetscan, miRanda, and miRBase programs, to elucidate the molecular mechanisms through which miR-205 functioned in human cervical carcinogenesis. Among the identified targets, CHN1 attracted our attention for following reasons: (i) there were two binding sites for miR-205 in the 3’UTR of CHN1, one of which was highly conserved among different species (Fig. 2a); and (ii) CHN1 has been reported to be a cancer-associated gene [26] involved in cell migration and cancer cell motility [24, 28]. CHN1 mRNA expression was elevated in the cervical cancer tissues than in the adjacent tissues of cancer (Fig. 1b). CHN1 protein expression was abnormally increased in tumour cell masses/cords, but was barely detectable or undetectable in normal cervical epithelium and noncancerous cervical stratified epithelium (Fig. 1a). Notably, in locations where miR-205 expression was enhanced, CHN1 was also increased; conversely, when miR-205 expression was decreased, CHN1 was also decreased. These data suggested that miR-205 and CHN1 were synchronously upregulated in human cervical tumours, which was contradictory to our expectations.

According to software-based prediction, the first seven nucleotides at the 5′-end of miR-205 were complementary to nucleotides 36–43 of the 3’UTR of the CHN1 gene (seed sequence; Fig. 2b). To validate whether CHN1 was a target of miR-205, HeLa cells were cotransfected with pmirGLO-CHN1-wt and miR-205 mimic or NC. Compared to the NC, transfection with the miR-205 mimic increased the relative luciferase activity of the pmirGLO-CHN1-wt construct (P < 0.05; Fig. 2b). Moreover, cotransfection of HeLa cells with a construct harbouring a mutated seed sequence and the NC or miR-205 mimic revealed that the miR-205 mimic could not increase the relative luciferase activity of the pmirGLO-CHN1-mut construct in the absence of the WT seed sequence (Fig. 2c). These results strongly supported that CHN1 was a target of miR-205 in HeLa cells.

As shown in Fig. 3a and b, the expression levels of miR-205 and CHN1 mRNA were higher in C33A cells and lower in SiHa cells than those in HeLa cells. The results of western blotting analysis of CHN1 protein were consistent with these results (Fig. 3c, Supplementary Figure 1). Thus, overall, these results demonstrated that high miR-205 expression was associated with high CHN1 expression, and vice versa, in the cervical cancer cell lines. The levels of miR-205, CHN1 mRNA, and CHN1 protein were significantly increased in cells transfected with miR-205 mimic compared with those in cells transfected with NC (Fig. 3d–f, Supplementary Figure 1). Conversely, the levels of miR-205, CHN1 mRNA, and CHN1 protein were reduced in cells transfected with miR-205 inhibitor, in comparison to those in cells transfected with inhibitor NC (Fig. 3g–i, Supplementary Figure 1). These results demonstrated that miR-205 positively and directly regulated the expression of CHN1 in human cervical cancer cells.

The functions of miR-205-dependent CHN1 expression in the pathological processes of cervical cancer cells were examined. At 48 h after transfection, compared to those in cells transfected with NC (Fig. 4a), the relative proliferation rates of HeLa, SiHa, and C33A cells transfected with miR-205 mimic were increased by about 13.38% (P < 0.05), 11.77% (P < 0.05), and 14.28% (P < 0.05), respectively. Conversely, the relative proliferation rates in HeLa, SiHa, and C33A cells transfected with miR-205 inhibitor were decreased by about 21.72% (P < 0.05), 17.56% (P < 0.05), and 18.62% (P < 0.05), respectively, in comparison with those in cells transfected with inhibitor NC (Fig. 4a). These results showed that overexpression of miR-205 significantly promoted cervical cancer cell proliferation, while downregulation of miR-205 suppressed cervical cancer cell proliferation.

Analysis of apoptosis revealed that transfection with the miR-205 mimic significantly increased early apoptosis in HeLa (P < 0.05) and SiHa cells (P < 0.05), but not in C33A cells (P > 0.05). Late apoptosis was also enhanced in HeLa (P < 0.01) and SiHa cells (P < 0.05), but not in C33A cells (P > 0.05), as compared with that in cells transfected with NC (Fig. 4b). Interestingly, transfection with miR-205 inhibitor did not affect early apoptosis in any of the three cell lines, while late apoptosis was increased in SiHa cells (P < 0.05), but not in HeLa or C33A cells (P > 0.05), as compared with those in cells transfected with inhibitor NC (Fig. 4b).

The migration capacity of HeLa, SiHa, and C33A cells transfected with the miR-205 mimic was significantly higher than that in cells transfected with NC (HeLa, P < 0.01; SiHa and C33A, P < 0.05; Fig. 5). Similarly, the migration capacity was decreased in HeLa, SiHa, and C33A cells transfected with miR-205 inhibitor, as compared with that in cells transfected with inhibitor NC (P < 0.05 for all cell lines; Fig. 5). Additionally, the invasive ability of HeLa and SiHa cells (HeLa, P < 0.05; SiHa, P < 0.01), transfected with the miR-205 mimic was evidently enhanced, but C33A cells (P > 0.05), as compared with that in cells transfected with the NC (Fig. 6). Conversely, transfection with the miR-205 inhibitor reduced invasive ability in HeLa and SiHa cells (HeLa and SiHa, P < 0.05), but not in C33A cells (P > 0.05), compared with that in cells transfect with the inhibitor NC (Fig. 6). These results suggested that miR-205 expression might be closely associated with the metastasis of cervical cancer cells.

To further examine whether miR-205 may have oncogenic roles through targeting of CHN1, the effects of CHN1 knockdown on miR-205-mediated cell behaviours were investigated. Importantly, transfection with si-CHN1 significantly downregulated CHN1 mRNA and protein expression (P < 0.05; Fig. 7a and b, Supplementary Figure 2). Functional analysis revealed that knockdown of CHN1 reduced cell viability, migration, and invasion (P < 0.05 for all) in SiHa cells (Fig. 7c and e). In addition, knockdown of CHN1 using si-CHN1 decreased the rate of apoptosis in SiHa cells (Fig. 7d). Finally, cotransfection of SiHa cells with si-CHN1 and the miR-205 mimic, resulted in lower capacity for proliferation, migration, invasion, and apoptosis (P < 0.05 for all) than those in cells transfected with the miR-205 mimic (Fig. 7c–e). These results demonstrated that the effects of miR-205 overexpression on cell growth and metastasis were partially attenuated by knockdown of CHN1.

The immunohistochemical analysis of CHN1 of 46 human cervical cancer samples were performed, to investigate the relationship between CHN1 overexpression and the clinical characteristics of human cervical cancer. The results showed that upregulation of CHN1 was not significantly associated with tumour size (P = 0.660), differentiation grade (P = 0.269), or depth of invasion (P = 0.962), suggesting that upregulation of CHN1 was a common characteristic in human cervical cancer (Table 1.). Therefore, CHN1 may be a novel biomarker for the clinical diagnosis of carcinogenesis of normal cervical epithelium.