Title: miR-375 Is Down-Regulated in Squamous Cervical Cancer and Inhibits Cell Migration and Invasion via Targeting Transcription Factor SP1
Abstract: Pelvic lymph node metastases are regarded as the most important risk factor and a predictor of poor prognosis for patients with cervical cancer. Exploration of metastasis-related molecules is helpful toward improving the prognosis in cervical cancer. To identify the role of miR-375 in metastasis and progression of cervical cancer, we examined the expression of miR-375 in 170 cervical cancer tissues and 68 normal cervical tissues, using stem-loop quantitative PCR, and found that the expression of miR-375 in cervical cancer tissues was significantly decreased by 4.45-fold, compared with 68 normal tissues. A significant correlation existed between miR-375 expression and clinicopathologic parameters, including lymph node metastasis of cervical cancer. Overexpressed miR-375 suppressed cell proliferation, blocked G1-to-S cell-cycle transition, and inhibited cell migration and invasion in human cervical SiHa and CaSki cells. SP1, a potential target gene of miR-375, was inversely correlated with miR-375 expression in cervical cancer tissues. Moreover, SP1 was negatively regulated by miR-375, and knockdown of SP1 by siRNA inhibited cell malignant behaviors. Thus, our findings suggest that down-regulated miR-375 promotes cell malignant behaviors via the target gene SP1 and may consequently contribute to the progression of cervical cancer. Pelvic lymph node metastases are regarded as the most important risk factor and a predictor of poor prognosis for patients with cervical cancer. Exploration of metastasis-related molecules is helpful toward improving the prognosis in cervical cancer. To identify the role of miR-375 in metastasis and progression of cervical cancer, we examined the expression of miR-375 in 170 cervical cancer tissues and 68 normal cervical tissues, using stem-loop quantitative PCR, and found that the expression of miR-375 in cervical cancer tissues was significantly decreased by 4.45-fold, compared with 68 normal tissues. A significant correlation existed between miR-375 expression and clinicopathologic parameters, including lymph node metastasis of cervical cancer. Overexpressed miR-375 suppressed cell proliferation, blocked G1-to-S cell-cycle transition, and inhibited cell migration and invasion in human cervical SiHa and CaSki cells. SP1, a potential target gene of miR-375, was inversely correlated with miR-375 expression in cervical cancer tissues. Moreover, SP1 was negatively regulated by miR-375, and knockdown of SP1 by siRNA inhibited cell malignant behaviors. Thus, our findings suggest that down-regulated miR-375 promotes cell malignant behaviors via the target gene SP1 and may consequently contribute to the progression of cervical cancer. Cervical cancer remains the second most common female malignant disease and is still one of the leading causes of cancer-related deaths worldwide. Although early-stage cervical cancer can be treated by radical surgery with or without chemotherapy and/or radiotherapy, some patients with high risk factors have an unfavorable prognosis.1Landoni F. Maneo A. Colombo A. Placa F. Milani R. Perego P. Favini G. Ferri L. Mangioni C. Randomised study of radical surgery versus radiotherapy for stage Ib-IIa cervical cancer.Lancet. 1997; 350: 535-540Abstract Full Text Full Text PDF PubMed Scopus (1375) Google Scholar, 2Biewenga P. van der Velden J. Mol B.W. Stalpers L.J. Schilthuis M.S. van der Steeg J.W. Burger M.P. Buist M.R. Prognostic model for survival in patients with early stage cervical cancer.Cancer. 2011; 117: 768-776Crossref PubMed Scopus (121) Google Scholar Various clinical data have shown that factors influencing prognosis in cervical cancer include tumor size, depth of stromal invasion, lymphovascular space involvement, parametrial invasion, and pelvic lymph node metastasis; of these, pelvic lymph node metastasis is regarded as the most important risk factor and predictor of poor prognosis for patients treated by surgery.3Potter M.E. Alvarez R.D. Shingleton H.M. Soong S.J. Hatch K.D. Early invasive cervical cancer with pelvic lymph node involvement: to complete or not to complete radical hysterectomy?.Gynecol Oncol. 1990; 37: 78-81Abstract Full Text PDF PubMed Scopus (50) Google Scholar The 5-year survival rate of patients with lymph node metastasis is diminished by 20% to 30%, compared with those without lymph node metastasis.4Shingleton H.M. Jones W.B. Russell A. Fremgen A. Chmiel J.S. Ocwieja K. Winchester D.P. Clive R. Hysterectomy in invasive cervical cancer: a national patterns of care study of the American College of Surgeons.J Am Coll Surg. 1996; 183: 393-400PubMed Google Scholar Thus, to identify molecular regulators associated with metastasis and to investigate their functions would likely contribute to improving the prognosis in cervical cancer. Some molecules have been identified, but their effect and mechanism are still uncertain. The finding of miRNAs provides a new approach and insight for exploring new molecules and mechanisms of metastasis and progression in cervical cancer. The miRNAs are a group of conserved noncoding RNAs that can bind to the 3′ untranslated region (UTR) of target mRNA and regulate stability and translation of mRNAs, resulting in either inhibition of translation or degradation of the target mRNA.5Bartel D.P. MicroRNAs: genomics, biogenesis, mechanism, and function.Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (29541) Google Scholar miRNAs can be classified as oncogenes or as tumor suppressors, and by targeting various transcripts they participate in diverse processes, including proliferation, apoptosis, metabolism, and cellular differentiation.6Lewis B.P. Shih I.H. Jones-Rhoades M.W. Bartel D.P. Burge C.B. Prediction of mammalian microRNA targets.Cell. 2003; 115: 787-798Abstract Full Text Full Text PDF PubMed Scopus (4222) Google Scholar Accumulated evidence has revealed that miRNAs are involved in metastasis and progression of various cancers, but their functions differ. For example, the overexpression of miR-3407Wu Z.S. Wu Q. Wang C.Q. Wang X.N. Huang J. Zhao J.J. Mao S.S. Zhang G.H. Xu X.C. Zhang N. miR-340 inhibition of breast cancer cell migration and invasion through targeting of oncoprotein c-Met.Cancer. 2011; 117: 2842-2852Crossref PubMed Scopus (181) Google Scholar and miR-218Yan L.X. Huang X.F. Shao Q. Huang M.Y. Deng L. Wu Q.L. Zeng Y.X. Shao J.Y. MicroRNA miR-21 overexpression in human breast cancer is associated with advanced clinical stage, lymph node metastasis and patient poor prognosis.RNA. 2008; 14: 2348-2360Crossref PubMed Scopus (974) Google Scholar promoted occurrence of lymph node metastasis, whereas the reduced expression of miR-10b and miR-3739Negrini M. Calin G.A. Breast cancer metastasis: a microRNA story.Breast Cancer Res. 2008; 10: 203Crossref PubMed Scopus (177) Google Scholar increased the risk of lymph node metastasis in breast carcinoma. To date, except for miR-127,10Lee J.W. Choi C.H. Choi J.J. Park Y.A. Kim S.J. Hwang S.Y. Kim W.Y. Kim T.J. Lee J.H. Kim B.G. Bae D.S. Altered MicroRNA expression in cervical carcinomas.Clin Cancer Res. 2008; 14: 2535-2542Crossref PubMed Scopus (281) Google Scholar few miRNAs have been reported to be associated with metastasis and progression in cervical cancer. In a previous study, we screened the different expression of miRNAs between cervical normal and cancer tissues through miRNA array and found that miR-375 was remarkably down-regulated in cervical cancer tissues, compared with normal tissues,11Li Y. Wang F. Xu J. Ye F. Shen Y. Zhou J. Lu W. Wan X. Ma D. Xie X. Progressive miRNA expression profiles in cervical carcinogenesis and identification of HPV-related target genes for miR-29.J Pathol. 2011; 224: 484-495Crossref PubMed Scopus (173) Google Scholar which implies that miR-375 may participate in the pathogenesis and development of cervical cancer. In the present study, we examined miR-375 expression in cervical cancer tissues and investigated the function of miR-375 in proliferation, migration, and invasion of cervical cancer cells, and we identified SP1 as a target gene for miR-375. miR-375 might act as a tumor suppressor and serve as a potential therapeutic target in cervical cancer. Cervical tissue samples and clinicopathologic data from 170 patients with primary cervical squamous cell carcinoma who underwent radical hysterectomy with pelvic lymph nodes dissection from July 2008 through December 2009 were collected in Women's Hospital, School of Medicine, Zhejiang University. Mean age was 44.8 years (range, 28 to 66 years). Of these 170 cervical cancer cases, 114 cases were International Federation of Gynecology and Obstetrics (FIGO) stage I and 56 were stage II; 29 cases were with lymph node metastasis and 141 without; 6 cases were well differentiated (G1), 148 were moderately differentiated (G2), and 16 were poorly differentiated (G3). No patient received chemotherapy or radiotherapy before the tissues were obtained. Normal cervical tissues with high-risk human papilloma virus (HR-HPV) infection (n = 32) or without HPV infection (n = 36) were collected as controls from women (mean age, 43.1 years; range, 27 to 64 years) who underwent hysterectomy for nonmalignant conditions. All histological diagnoses were made by two pathologists (Xiaoduan Chen and Bingjian Lv) independently. All specimens were immediately snap-frozen in liquid nitrogen and stored at −80°C until RNA extraction. The study was approved by the Ethics Committee of the hospital. RNA was isolated by the TRIzol method (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol, and the quantity and concentration of RNA were spectrophotometrically assessed by measuring absorbance at A260/280. Quantitative RT-PCR (qRT-PCR) for miRNA and mRNA was performed as described previously.11Li Y. Wang F. Xu J. Ye F. Shen Y. Zhou J. Lu W. Wan X. Ma D. Xie X. Progressive miRNA expression profiles in cervical carcinogenesis and identification of HPV-related target genes for miR-29.J Pathol. 2011; 224: 484-495Crossref PubMed Scopus (173) Google Scholar For miRNA quantification, each reverse transcript reaction consisted of 0.5 μg of total RNA, mixed with 2.0 μL of 5× RT buffer containing dNTPs (Takara Bio, Otsu, Japan), 0.2 μL of 10 μmol/L stem-loop RT primer (Invitrogen), 0.2 μL RNase inhibitor protein (Takara Bio), and 0.8 μL reverse transcriptase (Takara Bio) in a final volume of 10 μL, and incubated at 42°C for 60 minutes and at 85°C for 5 minutes. Real-time PCR was performed with an Applied Biosystems 7900HT system (Foster City, CA) using SYBR Premix Ex Taq (Takara Bio). PCR volume was 20 μL, containing 1 μL reverse transcript product. Cycling conditions were 1 cycle of 95°C for 30 s and 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds. PCR was performed in triplicate. For measurement of the SP1 transcript from total RNA, total cDNA was synthesized using a TaKaRa reverse transcription kit (Takara Bio). Real-time PCR was performed using SYBR Premix Ex Taq (Takara Bio). The U6 snRNA and EEF1A112Shen Y. Li Y. Ye F. Wang F. Lu W. Xie X. Identification of suitable reference genes for measurement of gene expression in human cervical tissues.Anal Biochem. 2010; 405: 224-229Crossref PubMed Scopus (55) Google Scholar were used as endogenous control for miRNA and mRNA, respectively. The sequences of all primers are given in Table 1. The ΔΔCt method was used to determine relative quantitation of miRNA and mRNA expression in tissue samples, and fold change was determined as 2−ΔΔCt.Table 1Sequences of Primers and siRNAPrimers and siRNASequencemiR-375 RT5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCACGC-3′miR-375 forward5′-AGCCGTTTGTTCGTTCGGCT-3′miR-375 reverse5′-GTGCAGGGTCCGAGGT-3′U6 snRNA RT5′-AACGCTTCACGAATTTGCGT-3′U6 snRNA forward5′-CTCGCTTCGGCAGCACA-3′U6 snRNA reverse5′-AACGCTTCACGAATTTGCGT-3′Sp1 forward5′-GGCTCGGGGGATCCTGGC-3′Sp1 reverse5′-TATGGCCCATATGTCTCTG-3′EEF1A1 forward5′-TGCGGTGGGTGTCATCAAA-3′EEF1A1 reverse5′-AAGAGTGGGGTGGCAGGTATTG-3′siRNA targeting Sp15′-ATCACTCCATGGATGAAATGA-3′Sp1 mUTR sense5′-GGAATGATAGCCCAGATGTAAAAAGAAATCTTGTCTTAC-3′Sp1 mUTR antisense5′-GTAAGACAAGATTTCTTTTTACATCTGGGCTATCATTCC-3′EEF1A1, eukaryotic translation elongation factor 1 alpha 1; mUTR: mutant untranslated region; RT, reverse transcriptase; siRNA, small interfering RNA; Sp1, Sp1 transcriptional factor. Open table in a new tab EEF1A1, eukaryotic translation elongation factor 1 alpha 1; mUTR: mutant untranslated region; RT, reverse transcriptase; siRNA, small interfering RNA; Sp1, Sp1 transcriptional factor. The HPV 16-positive human cervical carcinoma cell lines SiHa and CaSki were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2 in a humidified incubator. Cells were transfected with Dharmacon miRIDIAN miR-375 mimic (miR-375) and miRIDIAN microRNA mimic negative control 1 (negative control) (Thermo Fisher Scientific, Lafayette, CO) at a final concentration of 100 nmol/L, and small interfering RNA targeting SP1 (siR-SP1) and siR-Ribo negative control (siR-Cont) (RiboBio, Guangzhou, China) at a final concentration of 30 nmol/L, using DharmaFECT 1 transfection reagent (Thermo Fisher Scientific) in accordance with the manufacturer's instructions. The sequence of siRNA targeting SP1 is given in Table 1. The pmirGLO dual-luciferase miRNA target expression vector (pmirGLO vector) containing both firefly luciferase gene and Renilla luciferase gene was purchased from Promega (Madison, WI). Human SP1 3′UTR including the predicted binding site of miR-375 was amplified by RT-PCR (Table 1) and inserted into the 3′UTR region downstream of the firefly luciferase gene of pmirGLO vector (pmirGLO-UTR) using the XbaI and SacI restriction sites. A site-directed gene mutagenesis kit (Beyotime, Jiangsu, China) was used to construct the mutant type of miR-375-binding sites vector (pmirGLO-mUTR) according to the manufacturer's protocol. Both constructs were confirmed by restriction enzyme digestion and sequencing (Invitrogen). Cotransfection of miRNA mimics (50 nmol/L) and reporter vectors (0.2 μg/mL) was performed using DharmaFECT 1 transfection reagent. Luciferase activities were measured at 48 hours after transfection using a Dual-Glo luciferase assay system (Promega), and firefly luciferase activities were normalized to Renilla luciferase activities. For each comparison, values for cells with empty pmirGLO vector were set equally to 1. Experiments were performed in triplicate and repeated twice. Data are presented as means ± SD. At 72 hours after transfection with miR-375 mimics or siR-SP1, total protein extracts from the cells were prepared for Western blot analysis as described previously.11Li Y. Wang F. Xu J. Ye F. Shen Y. Zhou J. Lu W. Wan X. Ma D. Xie X. Progressive miRNA expression profiles in cervical carcinogenesis and identification of HPV-related target genes for miR-29.J Pathol. 2011; 224: 484-495Crossref PubMed Scopus (173) Google Scholar The primary antibodies used for Western blot analysis were anti-SP1 antibody (1:100) and anti-β actin antibody as an endogenous control (1:2000), both from Santa Cruz Biotechnology (Santa Cruz, CA). To determine the effect of miR-375 or siR-SP1 on proliferation of cervical cell lines, SiHa (5 × 103) and CaSki (4 × 103) cells were suspended in Dulbecco's modified Eagle's medium (100 μL) containing 10% fetal bovine serum and cultured in 96-well plates overnight and then transfected with miR-375 mimic or siR-SP1. The cell proliferation was determined with MTT assay at 24, 48, 72, and 96 hours after transfection respectively, and the absorbance of samples was measured with a spectrophotometer reader at 490 nm. All experiments were performed in six replicates and were repeated three times independently. Data are presented as means ± SD of three separate experiments. An in vitro wound-healing assay was used to assess capacity for tumor cell motility. Briefly, SiHa cells (5 × 105/well) and CaSki cells (5 × 105/well) were seeded in 12-well plates, cultured overnight, and transfected with miR-375 or siR-SP1. When the culture had reached nearly 90% confluency, the cell layer was scratched with a sterile plastic tip and then was immediately washed with growth medium twice and cultured again in Dulbecco's modified Eagle's medium (including 1% fetal bovine serum) at 37°C in a humidified incubator with 5% CO2 for up to 48 hours. At different time points, photographic images of the plates were acquired under a microscope. Wound healing was measured at 0, 24, and 48 hours, and the data were summarized based on six assays for each experiment. Invasion assay of SiHa and CaSki cells was examined in Transwell chambers with members coated with Cultrex Basement Membrane Extract without Phenol Red (R&D Systems, Minneapolis, MN). For this purpose, 2 × 104 cells transfected with miR-375 mimic or siR-SP1 were suspended in 200 μL Dulbecco's modified Eagle's medium without serum and seeded on the upper chamber; the lower chamber was filled with 10% fetal bovine serum as the chemoattractant. After 48 hours, cells on the upper side of the membrane were wiped off; cells on the lower side of the membrane were fixed and stained with crystal violet solution. The cells under the microscopic fields (100× objective) in each chamber were photographed and counted; values were expressed as fold induction. All invasion assays were done in triplicate for at least three independent experiments. Data are presented as means ± SD from at least three independent experiments. Student's t-test was used to analyze differences in experiments with cell lines. Correlation between expression levels of miR-375 and its target genes in cervical cancer tissues was analyzed using Pearson's correlation coefficient. Association between expression level of miR-375 and each clinicopathologic parameter was evaluated using Pearson's χ2 test. All statistical tests were two-sided, and P values of <0.05 were considered statistically significant. All analyses were performed using SPSS 16.0 software (SPSS, Chicago, IL). The expression of miR-375 was detected with real-time RT-PCR; U6 snRNA served as endogenous control. The relative quantitative analysis was calculated as 2-ΔΔCt. The 75th percentile of 2-ΔΔCt was used as a cut-off point for patients with high or low levels of miR-375, respectively.13Chen Z.L. Zhao X.H. Wang J.W. Li B.Z. Wang Z. Sun J. Tan F.W. Ding D.P. Xu X.H. Zhou F. Tan X.G. Hang J. Shi S.S. Feng X.L. He J. microRNA-92a promotes lymph node metastasis of human esophageal squamous cell carcinoma via E-cadherin.J Biol Chem. 2011; 286: 10725-10734Crossref PubMed Scopus (139) Google Scholar The mean relative miR-375 expression in 170 cervical cancer tissues was significantly decreased, by 4.45-fold, compared with that of 68 normal controls (P = 5.697 × 10−20) (Figure 1). In cervical cancer tissues, relative expression of miR-375 in tissues with lymph node metastasis was significantly lower than without the metastasis (χ2 = 6.263, P = 0.012). In addition, miR-375 expression was significantly correlated with other clinicopathologic parameters, including FIGO stage (χ2 = 9.394, P = 0.002), deep stromal invasion (χ2 = 9.392, P = 0.002), lymphovascular space involvement (χ2 = 4.453, P = 0.035), and vaginal wall extension (χ2 = 5.435, P = 0.020) (Table 2).Table 2Relations between Expression of miR-375 and Clinicopathologic Characteristics of Cervical Cancer TissuesCharacteristicNo.miR-375χ2P valueLowHighPatient age (years) ≤35221840.6760.411 >3514810939FIGO stage IB1/IB211477379.3940.002 IIA56506LNM Yes292726.2630.012 No14110041Differentiation well6420.2130.899 moderate14811137 poor16124Tumor size (cm) <4139100391.5920.207 ≥425214 undetermined642Stromal invasion <2/310570359.3920.002 ≥2/365578Vaginal wall extension Yes272525.4350.02 No14310241Parametrial extension Yes141220.9780.323 No15611541LVSI Yes584994.4530.035 No1127834FIGO, International Federation of Gynecology and Obstetrics; LNM, lymph node metastasis; LVSI, lymph vascular space invasion. Open table in a new tab FIGO, International Federation of Gynecology and Obstetrics; LNM, lymph node metastasis; LVSI, lymph vascular space invasion. SiHa and CaSki cells were transiently transfected with miR-375 mimic, which induced significant up-regulation of miR-375 compared with the negative control (see Supplemental Figure S1 at http://ajp.amjpathol.org). Cell proliferation was assayed by the MTT method. The proliferation rate of SiHa and CaSki cells at 96 hours after transfection was significantly reduced (P = 0.003 and P = 2.717 × 10−5, respectively), compared with that of miRNA negative control (Figure 2A). Analysis of cell cycle distribution by flow cytometry showed that the percentage of G1 phase of SiHa cells at 48 hours after transfection was significantly increased, from 63.4 ± 2.1% to 84.6 ± 2.9% (P = 6.197 × 10−4), and the percentage for S phase was significantly decreased, from 27.0 ± 1.1% to 8.6 ± 1.1% (P = 7.558 × 10−5). Similarly, the percentage of G1 of CaSki cells was significantly increased, from 66.4 ± 2.6% to 79.2 ± 4.6% (P = 1.354 × 10−5), and the percentage for S phase was significantly decreased, from 23.6 ± 1.6% to 12.0 ± 3.1% (P = 2.019 × 10−7) (Figure 2C). Thus, overexpression of miR-375 caused a significant G1 arrest, suggesting that elevated miR-375 blocks G1-to-S transition and consequently inhibits the progression of cell cycle in cervical cancer cells. The wound-healing assay showed that SiHa and CaSki cells with miR-375 overexpression using transient transfection with miR-375 mimic presented a slower closing of scratch wound, compared with the miRNA negative controls (P = 6.535 × 10−7 and P = 2.091 × 10−8, respectively) (Figure 3A). Moreover, Transwell invasion assay revealed that the invasion potential of SiHa and CaSki cells transfected with miR-375 was significantly decreased (P = 0.001 and P = 0.011, respectively), compared with miRNA negative control (Figure 4A). Our results indicate that miR-375 served as a tumor suppressor miRNA and contributed to inhibition of migration and invasion of cervical carcinoma cells.Figure 4Overexpression of miR-375 or knockdown of SP1 inhibited invasion of SiHa and CaSki cells. A: Overexpression of miR-375 inhibited invasion both in SiHa and CaSki cells. B: Knockdown of SP1 by siRNA inhibited invasion both in SiHa and CaSki cells. Data are presented as means ± SD from three independent experiments. *P < 0.05; **P < 0.01.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Two public algorithms and associated databases (TargetScan version 5.1, available at http://www.targetscan.org; PicTar, available at http://pictar.mdc-berlin.de) were used to find putative target genes for miR-375 that mediate cell growth, migration, and invasion. Gene Ontology analysis using the tools of AmiGO and CateGOrizer (GO; available at http://www.geneontology.org) was used to predict the genes common to both public databases (see Supplemental Figure S2 at http://ajp.amjpathol.org). The potential binding capability of SP1 3′UTR with miR-375 was predicted by both of the public algorithms (see Supplemental Figure S3 at http://ajp.amjpathol.org). SP1 was also an important gene with effective transcriptional regulation on various downstream genes involved in cell cycle, apoptosis, proliferation, and invasion. To confirm further that SP1 is a target gene for miR-375, RT-PCR and Western blot analysis were used to detect the expression of SP1 regulated by miR-375 in SiHa and CaSki. The expression of SP1 was significantly down-regulated after forced expression of miR-375: 37.6 ± 9.5% (P = 0.035) and 52.3 ± 5.9% (P = 0.010) at the mRNA level (Figure 5A) and 43.2 ± 9.1% (P = 0.023) and 49.2 ± 6.8% (P = 0.017) at the protein level (Figure 5, B and C), compared with miRNA negative control in SiHa and CaSki, respectively. Moreover, the mRNA levels of SP1 and miR-375 expression in one set of cervical cancer tissue were assessed by qRT-PCR. There was an inverse correlation between miR-375 and SP1 mRNA expression was found (Spearman's ρ = −0.437, P = 4.409 × 10−9). Taken together, our results together suggest that miR-375 negatively regulates SP1 gene expression and that SP1 is a potential target gene. To confirm that SP1 was a direct target of miR-375, pmirGLO-UTR containing the miR-375 binding sites was constructed to perform the reporter assay, and pmirGLO-mUTR containing mutant binding sites was used as a control. The luciferase activities of the reporter vectors were assayed at 48 hours after cotransfection with miR-375 mimic or negative control in SiHa cells. When cotransfected with miR-375, the relative luciferase activity of pmirGLO-UTR was significantly suppressed by 50% (P = 0.001), compared with cotransfection with negative control. Also, the relative luciferase activity was not altered when cotransfection was done with miR-375 and pmirGLO-mUTR containing a mutant binding site or empty pmirGLO (Figure 5G), indicating that SP1 was a direct target for miR-375. To investigate the role of SP1 in cell proliferation, migration, and invasion, siRNA targeting SP1 (siR-SP1) was used. Expression of SP1 was inhibited by 69.9 ± 7.2% (P = 0.008) and 52.4 ± 12.4% (P = 0.019) at the mRNA level (Figure 5D) and 70.2 ± 3.2% (P = 0.045) and 50.9 ± 8.4% (P = 0.017) at the protein level after knockdown of the SP1 gene in SiHa and CaSki cells (Figure 5, E and F). Consistently, the knockdown of SP1 markedly induced G1 arrest (P = 0.017 and P = 0.028) (Figure 2D) and inhibited cell proliferation (P = 9.315 × 10−7 and P = 3.905 × 10−4) (Figure 2B), migration (P = 3.738 × 10−4 and P = 0.002) (Figure 3B), and cell invasion (P = 0.013 and P = 0.027) (Figure 4B) in SiHa and CaSki, respectively, suggesting that up-regulated SP1 (induced by suppression of miR-375) participates in proliferation, migration, and invasion of cervical cancer cells and that knockdown of SP1 can reduce the malignant behaviors induced by diminished miR-375. To explore whether decreased miR-375 expression was induced by HPV oncoproteins, the expression of miR-375 was detected and compared in 32 normal cervical tissues with (n = 32) and without (n = 36) high-risk HPV infection. No difference of miR-375 relative expression was found between the two groups (P = 0.461). Furthermore, neither overexpression of HPV16 E6 by transfection with HPV-16 E6 expression vector14Li B. Hu Y. 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