MicroRNA-199a Inhibits Cell Proliferation, Migration, and Invasion and Activates AKT/mTOR Signaling Pathway by Targeting B7-H3 in Cervical Cancer.

Cervical cancer is a deadly disease. Some microRNAs are involved in tumor invasion and metastasis. Decreased expression of microRNA-199a has been correlated with tumorigenesis. In our study, the quantitative real-time polymerase chain reaction results indicated that microRNA-199a was expressed at lower levels in cervical cancer tissues, and the expression level of B7-H3 was significantly increased compared with that in the adjacent normal tissues, and the expression levels of B7-H3 and microRNA-199a in cervical cancer tissues and in adjacent normal tissues were inversely correlated. We also found that the expression of microRNA-199a was downregulated in cervical cancer cell lines when compared to immortalized cells. In this study, B7-H3 was identified as a novel target of microRNA-199a in cervical cancer. TargetScan (http://www.targetscan.org/) bioinformatics analysis was used to predict that the 3'-untranslated region of B7-H3 is a direct target of microRNA-199a. The result was also verified by the luciferase reporter assay. MicroRNA-199a could directly target the 3'-untranslated region of B7-H3, but the specific signaling pathways that were involved in regulating B7-H3 expression remained unclear. To clarify whether the suppressive effect of microRNA-199a was mediated through B7-H3, a series of experiments were performed. We found that the overexpression of microRNA-199a inhibited cell proliferation, migration, and invasion via direct binding to B7-H3. Epithelial-mesenchymal transition is a major factor involved in cervical cancer metastasis. Quantitative real-time polymerase chain reaction and western blot results indicated that microRNA-199a inhibits tumor progression in cervical cancer by targeting B7-H3. The microRNAs regulatory network is quite complex. We further examined the effect of microRNA-199a on the AKT/mTOR signaling pathway. We explored the regulatory role of microRNA-199a and first demonstrated that highly expressed microRNA-199a inhibits tumor growth and activates the AKT/mTOR signaling pathway by targeting B7-H3 in vivo and in vitro. Our findings not only provide a better understanding of the pathogenesis of cervical cancer but also provide novel findings and theoretical support for potential targeted therapeutic tools for cervical cancer.

Abbreviations: CC, cervical cancer; CCK-8, cell counting kit-8; cDNA, complementary DNA; EMT, epithelial–mesenchymal transition; FBS, fetal bovine serum; miRNA, microRNA; miR-NC, miR-negative control; miR-199a, microRNA-199a; mRNA, messenger RNA; NSCLC, non-small cell lung cancer; qRT-PCR, quantitative real-time polymerase chain reaction; 3′-UTR, 3′-untranslated region.

Introduction

Cervical cancer (CC) is one of the most common malignancies in females[1] and its incidence ranks fifth in China with nearly 1 × 104 new cases and 3 × 104 deaths in 2015.[2] Although the therapeutic efficacy of treatments for early CC is improving, the overall 5-year survival rate for advanced-stage CC is still very low.[3-5]

B7-H3 is a member of the B7 family of proteins. An increasing number of studies have shown that B7-H3 is highly expressed in carcinomas of the colon,[6] gastric cancer,[7] breast cancer,[8] and lung cancer,[9] but it is not expressed or is expressed at low levels in most normal cells and tissues.[5,10] MicroRNAs (miRNAs) are of great importance in major biological processes and play a crucial role in the regulation of development, proliferation, differentiation migration, and invasion due to their regulation of target gene expression.[11] MicroRNA-199a (miR-199a), a member of the most important miRNA family, is involved in a variety of cancers and serves different functions in different cancers. Studies have found that miR-199a modulates the inflammatory microenvironment in ovarian cancer and alleviates invasion through the Akt/mTOR signaling in breast cancer.[10,12,13] In gastric cancer, miR-199a is highly expressed and promotes proliferation, migration, and invasion.[14] In colorectal cancer, miR-199a can inhibit the proliferation, migration, and invasion.[15] In breast cancer and CC, miR-199a is significantly decreased.[16,17] Several studies have reported that miRNAs regulate tumor progression by targeting B7-H3 in breast cancer, osteosarcoma, and renal cell carcinoma.[18-20] Moreover, studies have found that miR-29, miR-187, miR-124, and miR-155/143 can regulate the tumor microenvironment by targeting B7-H3, and participate in the regulation of tumorigenesis and development by binding to the 3′-untranslated region (3′-UTR) of B7-H3.[20-22] However, the precise mechanisms by which miR-199a regulate B7-H3 are unclear. Our study aimed to investigate the regulatory role of miR-199a and first demonstrated that highly expressed miR-199a inhibits tumor growth and activates the AKT/mTOR signaling pathway by targeting B7-H3 in vivo and in vitro.

Methods

Clinical Samples

A total of 30 pairs of CC tumor and matched nontumor tissues were collected at the Central Hospital of Panyu District between February 2016 and January 2019. All patients provided written informed consent for tissue sample analysis. Fresh CC tissues and matched cancer-adjacent tissues were sampled directly after surgical removal, and immediately frozen in liquid nitrogen for further use. All patients were well informed and the process was approved by Ethics Committee of Central Hospital of Panyu District, Guangdong, China.

Cell Cultures and Reagents

Cervical cancer cell lines were purchased from the American Type Culture Collection. The following primary antibodies were purchased from Cell Signaling Technology, Inc: E-cadherin (#3195S), N-cadherin (#5741S), B7-H3 (#14058S), AKT (#9272S), p-AKT (#4060S), and mTOR (#2972S). GAPDH (#sc-47724) was purchased from Santa Cruz Biotechnology. The data were collected from at least 3 independent experiments.

Cell Proliferation Assay

Cell proliferation activity was analyzed with a cell counting kit-8 (CCK-8) (Dojindo) following the manufacturer’s instructions. The inhibition rate of the cell proliferation was calculated for each well as (A450 control cells – A450-treated cells)/A450 control cells × 100%. Experiments were performed in triplicate.

Luciferase Reporter Assays

Cells were cotransfected with B7-H3 wt/mut 3′-UTR plasmids (Invitrogen) and either the miR-199a-mimics or miR-negative control (miR-NC; RiboBio) using Lipofectamine 3000 (Invitrogen). After 48 hours, Luciferase activity was assessed in the indicated cells using the Luciferase Reporter Assay System (Promega), according to the manufacturer’s instructions. The experiments were replicated in triplicate.

Enzyme-linked immunosorbent assay

Enzyme-linked immunosorbent assay Kit (Sino Biological Inc) were used for human B7-H3 detection, according to the manufacturer’s instructions.

Western Blot Assays

Cells were lysed in lysis buffer and tumors were lysed in RIPA buffer. After incubating on ice for 30 minutes, the cells were centrifuged at 12 000g for 15 minutes at 4 °C, and the supernatant was collected. Samples were then analyzed by western blotting. Proteins were visualized by incubation with SuperSignal West Pico reagents (NCI5079; Thermo), followed by exposure to radiographic film.

Quantitative Real-Time Polymerase Chain Reaction

Total RNA was extracted from 30 pairs of frozen tissue samples and cells, using TRIzol (Invitrogen) according to the manufacturer’s protocol. First-strand complementary DNA (cDNA) from RNA templates was generated using a RevertAid First-Strand cDNA Synthesis Kit (Fermentas) according to the manufacturer’s protocol. The primers used in a quantitative real-time polymerase chain reaction (qRT-PCR) were designed by GenePharma and the primer sequences were as follows: miR-199a: (F) 5′-ACACTCCAGCTGGGAGTGTTCAGACTA-3′ and (R) 3′-TGTGCCCCTAACCCTTTC-5′ and human B7-H3: (F) 5′-TGCACCACCAACTGCTTAGC-3′ and (R) 3′-GGCATGGACTGTGGTCATGAG-5′. The results of U6 qRT-PCR gene expression analysis with the 2−ΔΔCt method were used as the control. All of the real-time PCR assays were performed in triplicate.

Cell Migration and Invasion Assays

The migratory and invasive ability of CC cells were determined by transwell assays. Transwell chamber (8.0 mm PC, Corning-Costar) inserts uncoated or coated with Matrigel (BD Biosciences) were used for cell migration and invasion assays, respectively. Cells were resuspended in 100 µL of the corresponding culture medium without fetal bovine serum (FBS) and were loaded into the top chamber of the transwell insert with a noncoated membrane. The bottom chamber contained 600 mL of medium with 20% FBS. Cells were then allowed to migrate at 37 °C, and the cells that left on the upper chamber were removed with a cotton swab. The filter was fixed with 95% ethanol for 20 minutes and then stained with 4 g/L crystal violet for 30 minutes. Cells were photographed in 5 independent 20× magnification fields under an inverted microscope (Nikon) and were counted. All experiments were independently repeated for at least 3 times.

Immunohistochemical Staining

Immunohistochemical staining was performed to detect the B7H3 expression level in tissues from patients with CC and that in adjacent tissues. The paraffin-embedded sections were stained with antibodies against B7H3 for 1 hour at dilution 1:100. The slides were analyzed by standard light microscopy. The negative control was stained with immunoglobulin G as the primary antibody.

Nude Mouse Xenograft Studies

Four-week-old BALB/c (athymic) nude mice were maintained under pathogen-free conditions. A total of 3 × 106 cells (cells were transfected with miR-199a-mimics or miR-NC) were subcutaneously injected into the right flank of nude mice. Body weights and tumor volumes (V) were measured every 2 days. Tumor volumes were calculated according to the formula: V = (length × width[2])/2. At 25 days postinjection, the mice were sacrificed and tumors from each mouse were removed for the following experiments.

Statistical Analysis

All assays were performed in triplicate. Data are expressed as the mean ± standard deviation. One-way analysis of variance was performed for multiple comparisons using GraphPad Prism software, version 5.0 (GraphPad). Value of P ≤ .05 indicated a statistically significant difference.

Results

The Expression Levels of miR-199a and B7-H3 Were Inversely Correlated in CC Tissues and Cells

First, the expression pattern of miR-199a was analyzed in CC tissues and adjacent normal tissues. The qRT-PCR results indicated that miR-199a was expressed at lower levels in CC tissues than in adjacent normal tissues (Figure 1A). In addition, the immunohistochemistry staining showed that the expression level of B7-H3 was significantly upregulated in CC tissues compared with that in the adjacent tissues (Figure 1B, C). Then, Spearman rank-order correlation analysis was used to investigate the relationship between miR-199a and B7-H3 in patients with CC. As shown in Figure 1D, the expression levels of B7-H3 and miR-199a were inversely correlated (R 2 = 0.5089, P = .0029). To analyze the mechanism by which miR-199a affects the biological functions of CC cells (CC cell lines: HeLa/C4-1/SiHa/CaSki/C-33A; Immortalized cells: Ect1/E6E7). Our analysis revealed that miR-199a was downregulated in CC cells compared to the immortalized cells (Figure 1E). These results support our hypothesis that the expression of miR-199a and B7-H3 might be involved in the progression of CC and that the expression levels of B7-H3 and miR-199a were inversely correlated.

Figure 1.

The expression of miR-199a and B7-H3 in CC tissues and cells. A, The expression of miR-199a in CC tissues and adjacent normal tissues. B, Immunohistochemical (IHC) staining of B7H3 (representative picture). C, Quantification of IHC of B7H3. D, The relationship between miR-199a and B7-H3 in patients with CC. E, The expression of miR-199a in CC cells. Error bars represent the standard deviation. ***P < .001 versus Ect1/E6E7 cells. CC indicates cervical cancer; miR, microRNA.

The 3′-UTR of B7-H3 Messenger RNA Is a Direct Target of miR-199a

To analyze the biological role of miR-199a and B7-H3, TargetScan (http://www.targetscan.org/) bioinformatics analysis was used to predict that the 3′-UTR of B7-H3 is a direct target of miR-199a (Figure 2A, B). Furthermore, all recombinant lentiviruses: miR-199a mimics and miR-NC were transfected into CC cells. Real-time polymerase chain reaction results (Figure 2C) showed that the miR-199a expression levels were markedly upregulated in the miR-199a-mimic groups compared to the miR-NC groups (P < .01). In addition, the messenger RNA (mRNA) and protein expression levels of B7-H3 are shown in Figure 2D and E, and the expression of B7-H3 was significantly downregulated when the expression of miR-199a was upregulated. To further confirm our result, the B7-H3 wt/mut 3′-UTR was cloned into the pGL3-control vector (Figure 2F). Then the B7-H3 wt/mut vector were cotransfected into CC cells, and a dual-luciferase assay was performed. The results showed that miR-199a-mimics reduced the luciferase activity of the wt B7-H3 3′-UTR reporter construct (P < .05). However, no difference was observed in the luciferase activity of the other groups. In summary, the results suggest that the 3′-UTR of B7-H3 mRNA is a direct target of miR-199a in CC cells.

Figure 2.

MiR-199a downregulates the expression of B7-H3 by directly targeting the B7-H3 3′-UTR. A, The predicted sites of miR-199a binding to the 3′-UTR of B7-H3 were identified using bioinformatics prediction tools. B, The mutated site in the 3′-UTR of B7-H3 is also shown. C, Cells were infected with lentivirus, and the expression level of miR-199a in the miR-199a-mimics groups and miR-NC groups was measured by qRT-PCR. **P < .01. D, The expression level of B7-H3 in the miR-199a-mimics groups and miR-NC groups, as measured by qRT-PCR. **P < .01. E, The protein level of B7-H3 in cells. F, SiHa cells were cotransfected with the miR-199a-mimics or miR-NC and the B7-H3 wt 3′-UTR reporter plasmid or its mutant form. The Luciferase activity was detected 48 hours after transfection. The data are shown as the mean ± standard deviation of 3 replicates (**P < .01). miR-199a indicates microRNA-199a; miR-NC, miR-negative control; qRT-PCR, quantitative real-time polymerase chain reaction; 3′-UTR, 3′-untranslated region.

MiR-199a Inhibits Cell Proliferation of CC Cells by Targeting B7-H3

To further confirm the mechanism of the cellular biological function of miR-199a and B7-H3, CC cells were cotransfected with an empty vector (Empty) or a vector carrying B7-H3 (B7H3) and miR-NC or miR-199a-mimics. Western blotting was used to detect the expression of B7-H3, as shown in Figure 3A and B. Compared with that in the miR-NC/Ep groups, the expression of B7-H3 in the miR-199a/Ep groups was significantly downregulated, and the expression of B7-H3 in the miR-199a/B7H3 was partially reversed by B7-H3 overexpression. Then, CCK-8 assays were conducted to assess the effect of miR-199a on CC cell proliferation. We examined the viability of the cells at 0, 24, 48, 72, and 96 hours, respectively (P < .05, Figure 2C, D). The results showed that compared to that of the miR-NC/Ep groups, cell viability of the miR-199a/Ep groups was suppressed, cell viability of the miR-NC/B7H3 groups was increased, and cell viability of the miR-199a/B7H3 groups was partially reversed. Our data strongly suggest that the overexpression of miR-199a inhibits cell proliferation via direct binding to B7-H3.

Figure 3.

MiR-199a overexpression inhibits the proliferation of CC cells by B7-H3 in vitro. SiHa and HeLa cells were treated with miR-NC and Empty (miR-NC/Ep), miR-NC and B7-H3 (miR-NC/B7H3), miR-199a-mimics and Empty (miR-199a/Ep), miR-199a-mimics and B7-H3 (miR-199a/B7H3). A and B, B7-H3 mRNA and protein levels in the cells. C and D, Effects of miR-199a on the proliferation of cells. A CCK-8 assay was performed at 0, 24, 48, 72, and 96 hours. The absorbance at 450 nm was measured. The data are presented as the mean ± standard error of at least 3 independent experiments. ***P < .001. CC indicates cervical cancer; CCK-8, cell counting kit-8; miR-199a, microRNA-199a; miR-NC, miR-negative control.

MiRNA-199a Inhibits the Migration and Invasion of CC Cells by Targeting B7-H3

To further confirm that the tumor suppressive roles of miR-199a were mediated by B7-H3, cell migration and invasion assays were performed. As shown in Figure 3A-C, the overexpression of B7-H3 significantly promoted migration (Figure 4A, B) and invasion (Figure 4C), miR-199a-mimics were significantly inhibited migration and invasion, and the effects of miR-199a-mimics were partially reversed by overexpression of B7-H3.

Figure 4.

MicroRNA-199a suppresses the migration and invasion of CC cells by targeting B7-H3 in vitro. SiHa and HeLa cells were cotransfected with miR-199a-mimics or miR-NC, and the B7-H3 wt plasmid or the empty plasmid. A and B, The migration abilities of cells on the membrane ± SE. **P < .01. C, Bar graphs of the invasion abilities of CC cells on the underside of the membrane ± SE. **P < .01. D, The mRNA expression levels of EMT markers (E-cadherin, N-cadherin, Vimentin, Snail, and Slug) were assessed using qRT-PCR. E, The protein levels of EMT markers (E-cadherin and N-cadherin) were assessed using western blotting. The data are presented as the mean ± SE of at least 3 independent experiments. **P < .01. CC indicates cervical cancer; EMT, epithelial–mesenchymal transition; miR-199a, microRNA-199a; miR-NC, miR-negative control; mRNA, messenger RNA; qRT-PCR, quantitative real-time polymerase chain reaction; SE, standard error.

Epithelial–mesenchymal transition (EMT) is a major factor involved in CC metastasis. Thus, qRT-PCR was conducted to investigate the expression levels of EMT factors relative to the GAPDH. Then, western blotting was used to analyze the expression levels of E-cadherin and N-cadherin. The qRT-PCR (Figure 4D) and western blot (Figure 4E) results were consistent with the previous results. Taken together, these observations indicate that miR-199a inhibits tumor progression of CC by targeting B7-H3.

Highly Expressed miR-199a Affects the AKT/mTOR Signaling Pathway by Targeting B7-H3

The regulatory network of miRNAs is quite complex. We further examined the effect of miR-199a on the AKT/mTOR signaling pathway. SiHa and HeLa cells were cotransfected with the miR-199a-mimics or miR-NC and the B7-H3 wt plasmid or the empty plasmid. Figure 5A-D shows that compared to that in the miR-NC groups, the expression of p-mTOR and p-AKT in the miR-NC-B7-H3 groups was decreased, and the expression of p-mTOR and p-AKT in the miR-199a-mimics groups was increased. When miR-199a-mimics and B7-H3 were coexpressed, the downregulated expression of p-mTOR and p-AKT was partially reversed. Consistent with the above results, these results indicate that the overexpression of miR-199a affects the AKT/mTOR signaling pathway by targeting B7-H3.

Figure 5.

The overexpression of miR-199a affects the AKT/mTOR signaling pathway. (A and B) Western blot and (C and D) qPCR analyses of the AKT/mTOR signaling pathways in CC cells were cotransfected with miR-199a mimics or miR-NC, and the B7-H3 wt plasmid or the empty plasmid. CC indicates cervical cancer; miR-199a, microRNA-199a; miR-NC, miR-negative control; qPCR, quantitative polymerase chain reaction.

MiR-199a Suppresses Tumor Growth In Vivo

To further investigate the role of miR-199a in vivo, we utilized a nude mouse xenograft model. It was found that miR-199a-mimics significantly inhibited tumor growth in mice (Figure 6A and B). Moreover, tumor weights were reduced in the miR-199a-mimics group (Figure 6C). Notably, the expression of miR-199a in the tumor tissues was increased compared to that in the miRNA-NC group. In addition, we examined the expression of miR-199a and B7-H3 in mouse tumor tissues. Consistent with the in vitro results, the expression levels of B7-H3, p-AKT, and p-mTOR were markedly downregulated in the miR-199a-mimics group xenografts (Figure 6D). Thus, miR-199a inhibits tumor growth in vivo. Taken together, these results suggest that the overexpression of miR-199a inhibits tumor growth of CC in vitro and in vivo and activates the AKT/mTOR signaling pathway through B7-H3.

Figure 6.

MicroRNA-199a suppresses tumorigenesis in nude mice. A-C, Changes in the tumor volume and tumor growth between the miR-199a-mimics group and the miR-NC group of CC tumor-bearing mice. Data are shown as the mean tumor volume ± SD (8 mice/group). ***P < .001. D, The expression level of miR-199a in tumor tissues. ***P < .001. E, The expression levels of B7-H3, AKT, p-AKT, mTOR, and p-mTOR were analyzed by Western blotting. CC indicates cervical cancer; miR-199a, microRNA-199a; miR-NC, miR-negative control; SD, standard deviation.

Discussion

Cervical cancer is the most common malignancy in women worldwide. MiR-dysregulation is associated with a wide variety of human malignancies. The role of miRNAs in various cancers has attracted increasing attention, and this provides new insights into the molecular mechanisms and therapeutic strategies of cancer. Studies have revealed that many miRNAs, such as miR-18a, miR-132, and miR-145, participate in the biological responses of CC.[23-25] The downregulation of miR-199a was observed in hepatocellular carcinoma,[26] non-small cell lung cancer (NSCLC),[27] and pancreatic tumors.[28] In our study, the qRT-PCR results indicated that miR-199a was expressed at lower levels in CC tissues than in normal tissues.

Recent studies have shown that B7-H3 is upregulated in various malignant tumors including pancreatic cancer,[29] prostate cancer,[30] renal cell carcinoma,[31,32] and NSCLC,[33] but it is widely expressed at low levels in normal tissues.[34] In addition, Huang et al [28] found that high expression of B7-H3 was significantly correlated with tumor size and prognosis in patients with CC. Our results indicated that the expression level of B7-H3 was significantly increased compared with that in the adjacent tissues. B7-H3 plays an important role in promoting tumor cell proliferation and metastasis, similar to downregulated miR-199a. However, the expression patterns and roles of B7-H3 and miR-199a in CC have not yet been investigated. Our research shows that the expression of miR-199a and B7-H3 might be involved in the progression of CC and that the expression levels of B7-H3 and miR-199a were inversely correlated. We also found that the expression of miR-199a was downregulated in CC cell lines compared to the mortalized cells. Furthermore, it has been reported that B7-H3 participates in miRNA-related regulatory processes.[21] TargetScan (http://www.targetscan.org/) bioinformatics analysis was used to predict the 3′-UTR of B7-H3 is a direct target of miR-199a. In addition, this result was validated in the luciferase reporter assay. MiR-199a could directly target the B7-H3 3′UTR, but the specific signaling pathways that were involved in regulating B7-H3 expression remained unclear. In further research, we successfully constructed miR-199a-mimics and miR-NC lentiviral expression vectors, creating the basis for a subsequent study on the function of miR-199a. Then, we explored the biological role of miR-199a at the cellular level. Cell counting kit-8 assays were conducted to assess the effect of miR-199a on cell proliferation, migration, and invasion. Our data strongly suggest that the overexpression of miR-199a inhibits cell proliferation, migration, and invasion via direct binding to B7-H3. Epithelial–mesenchymal transition is a major factor involved in CC metastasis. Here, qRT-PCR and western blot results indicate that miR-199a inhibits tumor progression in CC by targeting B7-H3.

The regulatory network of miRNAs is quite complex. Studies have shown that miR-199a not only inhibits the aggressiveness of cancer cells through the AKT/mTOR signaling pathway in human liver cancer[35,36] but also regulates the migration and invasion of breast cancer.[10] In this study, we explored the regulatory role of miR-199a and first demonstrated that highly expressed miR-199a inhibits tumor growth in vivo and in vitro and activates the AKT/mTOR signaling pathway by targeting B7-H3 in CC cells.

Our findings not only provide a better understanding of the pathogenesis in CC and but also provide novel findings and theoretical support for potentially targeted therapeutic tools for CC, it also provides theoretical support for potentially targeted therapies for CC. In conclusion, our results suggest that miR-199a acts as a potential mechanism for CC development by directly targeting B7-H3.