• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    Aberrant methylation frequency of TNFRSF10C promoter in pancreatic cancer cell lines

    2011-07-07 01:00:09HuiHuaCaiYueMingSunYiMiaoWenTaoGaoQuanPengJieYaoandHanLinZhao
    關鍵詞:科研成果性質形式

    Hui-Hua Cai, Yue-Ming Sun, Yi Miao, Wen-Tao Gao, Quan Peng, Jie Yao and Han-Lin Zhao

    Nanjing, China

    Aberrant methylation frequency of TNFRSF10C promoter in pancreatic cancer cell lines

    Hui-Hua Cai, Yue-Ming Sun, Yi Miao, Wen-Tao Gao, Quan Peng, Jie Yao and Han-Lin Zhao

    Nanjing, China

    BACKGROUND:A growing body of evidence suggests that many tumors are initiated by both epigenetic abnormalities and gene mutations, which promote tumor progression. Epigenetic abnormalities include changes in DNA methylation and in the modification of histones. This study aimed to assess the status of methylation in the CpG island (CGI) of the tumor necrosis factor receptor superfamily member 10c (TNFRSF10C) with combined bisulfite restriction analysis (COBRA) and to evaluate its role in the progression of pancreatic cancer (PC).

    METHODS:The methylation status of four PC cell lines was assessed using COBRA and/or bisulfite genomic sequencing (BGS). Changes in methylation and TNFRSF10C expression in PC cell lines before and after treatment with 5-aza-2'-deoxycytidine (5-aza-dC) and/or trichostatin A (TSA) were assessed by BGS and real-time RT-PCR. Apoptosis in the four cell lines was tested by flow cytometry (FCM) and TUNEL assay.

    RESULTS:The methylation status of the TNFRSF10C promoter was assessed in PC cells (BxPC-3: 68.84±8.71%; CFPAC-1: 0; PANC-1: 96.77±4.57%; SW1990: 54.97±7.33%) with the COBRA assay, which was confirmed by the results of BGS. After treatment with 5-aza-dC and/or TSA, apoptosis was induced in PC cells to different degrees, and the levels of TNFRSF10C transcriptional expression in the PC cell lines (except CFPAC-1) increased markedly after 5-aza-dC treatment.

    CONCLUSIONS:A high frequency of CGI methylation in the TNFRSF10C promoter results in inactivation of the gene and enhancement of tumor growth in most PC celllines (except CFPAC-1). Inactivation of TNFRSF10C by CGI hypermethylation can play an important role in PC progression and be potentially useful as a diagnostic marker and a new therapeutic approach for PC.

    (Hepatobiliary Pancreat Dis Int 2011; 10: 95-100)

    methylation; CpG island; pancreatic cancer; TNFRSF10C

    Introduction

    Pancreatic cancer (PC) is a malignant tumor associated with a significant degree of morbidity and mortality.[1]It is highly invasive and responds poorly to conventional treatments. Like other tumor types, PC arises from a complex and poorly understood sequence of genetic and epigenetic alterations. Aberrant hypermethylation of CpG islands (CGIs) in gene promoters and histone deacetylation are the main epigenetic changes that lead to gene silencing, but this has not been thoroughly investigated in PC cell lines. Elucidating the mechanisms of these regulatory events is likely to enhance our understanding of this disease and the identification of new treatment targets.

    The tumor necrosis factor receptor superfamily member 10c (TNFRSF10C) is one of several TNF-related, apoptosis-inducing ligand (TRAIL)-like decoy receptors. It is a glycosyl phosphatidylinositol-linked membrane molecule that lacks a cytoplasmic region and is located on 8p22-p21.[2]TNFRSF10C is one of the most frequently deleted loci in cancers of the colon,[3]lung,[4]prostate,[5,6]breast[7,8]and bladder.[9]Hypermethylation, specifically of the CGI in the TNFRSF10C promoter, has been reported in neuroblastoma (21%),[10]primary breast cancer (48%), primary lung cancer (37%), malignant mesothelioma (43%),[8]ependymoma (50%), and choroid plexus papilloma (50%).[11]Furthermore, a recent study by Hornstein et al[12]reported a significant decrease in expression of TNFRSF10C in the M0 stage of prostate carcinoma tissue compared to benign prostate tissue.These data suggest that TNFRSF10C is a tumor suppressor gene of prostate carcinoma. However, hypermethylation of the TNFRSF10C promoter in PC has not previously been investigated.

    In the present study, we determined the frequency of hypermethylation in the TNFRSF10C promoter in four PC cell lines (BxPC-3, CFPAC-1, PANC-1, and SW1990) by using combined bisulfite restriction analysis (COBRA) and/or bisulfite genomic sequencing (BGS). Changes in methylation status and gene expression of TNFRSF10C before and after treatment with 5-aza-2'-deoxycytidine (5-aza-dC) or trichostatin A (TSA) were also evaluated in these PC cell lines.

    Methods

    Cell culture and drug treatment

    The PC cell lines (BxPC-3, CFPAC-1, PANC-1, and SW1990) were cultured in DMEM (Hyclone, Logan, UT), and all cells were maintained at 37 ℃ and 5% CO2in a humidified incubator. Treatments of cell lines were: 100 nmol/L TSA (Sigma, St. Louis, MO) for 24 hours, 5 μmol/L 5-aza-dC (Sigma, St. Louis, MO) for 72 hours (with fresh drug added every 24 hours), and 5 μmol/L 5-aza-dC for 72 hours followed by 24 hours with 100 nmol/L TSA.[13]The control group was untreated. Apoptosis before and after drug treatment was assayed using flow cytometry (FCM) and TUNEL assay.

    Isolation of RNA and genomic DNA

    RNA from cells was isolated using Trizol according to the manufacturer's instructions. Recovered RNA was further treated with DNaseiand re-purified using a Micro-to-Midi total RNA purification system (Invitrogen, Carlsbad, CA, USA).

    Genomic DNA from cultured cells was isolated from the inter-organic phase of a Trizol-chloroform mixture, then combined with 0.5 ml of back extraction buffer (4 mol/L guanidine thiocyanate, 50 mmol/L sodium citrate, 1 mol/L Tris, pH 8.0). After vigorous mixing by inversion, phase separation was achieved by centrifugation at 12 000×g for 30 minutes. DNA was precipitated from the upper aqueous phase using isopropanol and further purified using Gentra Puregene reagent (Qiagen, Hilden, Germany) with proteinase K treatment.[14]

    Bisulfite modification

    DNA (4 μg) was denatured with 0.2 mol/L NaOH for 20 minutes at 37 ℃ and treated with sodium bisulfite (Sigma, St Louis, MO) for 16 hours at 50 ℃. Modified DNA was purified using the Wizard DNA Clean-up system (Promega, Madison, WI) and diluted in 50 μl nuclease-free water (NFH2O). Modification was completed by incubation with NaOH (0.3 mol/L final concentration) for 5 minutes at room temperature. DNA was precipitated with ethanol and resuspended in 60 μl NFH2O. DNA from SW1990 cells was treated with CpG methyltransferase (M.SssI) (NEB, Ipswich, MA) according to the manufacturer's protocol and modified by sodium bisulfite for a positive control.

    BGS

    TNFRSF10C methyl primers were designed using Methyl-Primer-Express software v1.0 (Applied Biosystems, Foster City, CA) to amplify bisulfite-modified DNA. The forward and reverse primer sequences were: 5'-GGA TCC CCA AGA CCC TAA AG-3' and 5'-TGT TGG AAG CGT TGG TGT AA-3', respectively, and a 215-bp product amplified contained 10 CpG dinucleotides. PCR samples contained 36.3 μl NFH2O, 5 μl 10×buffer (Mg2+), 2.5 μl 10 mmol/L dNTP, 2 μl of each primer, 2 μl bisulfite-treated DNA, and 0.2 μl FastTaqTMpolymerase (Qiagen, Hilden, Germany). Amplification was performed with the following cycles: (a) 95 ℃ for 5 minutes; (b) 2 cycles of 95 ℃for 45 seconds, 68 ℃ for 30 seconds, and 72 ℃ for 30 seconds; (c) 3 cycles of 95 ℃ for 45 seconds, 66 ℃ for 30 seconds, and 72 ℃ for 30 seconds; (d) 4 cycles of 95 ℃ for 45 seconds, 64 ℃ for 30 seconds, and 72 ℃for 30 seconds; (e) 5 cycles of 95 ℃ for 45 seconds, 62 ℃ for 30 seconds, and 72 ℃ for 30 seconds; (f) 35 cycles of 95 ℃ for 45 seconds, 60 ℃ for 30 seconds, and 72 ℃ for 30 seconds; with a final extension step for 7 minutes at 72 ℃. Amplified PCR products (5 μl) were separated by electrophoresis in 2% agarose gels and visualized by ethidium bromide staining. Using the Toyobo TA cloning kit (Toyobo, Osaka, Japan), 2 μl of PCR product was subcloned into the pCR4-TOPO vector and transformed into E. coli. Sequencing analysis was performed on 10-20 randomly-selected individual colonies and the data were compared with the UCSC genome reference sequence to assess the methylation status of each CpG site.

    COBRA[15]

    The restriction enzyme TaqI (NEB, Ipswich, MA) was included in COBRA assay samples as follows: 18 μl NFH2O, 2 μl 10×buffer, 10 μl PCR product, and 2 μl TaqI. Reaction conditions were (a) 65 ℃ for 16 hours and (b) 80 ℃ for 20 minutes. Digested PCR products were separated on an 8% polyacrylamide gel, visualized by ethidium bromide staining, and photographed usinga Bio-Rad gel documentation system. Quantitation was performed using a Molecular Dynamics phosphor imager.

    Quantitative RT-PCR

    Total RNA was purified from cultured cells before and after treatment with 5-aza-dC or TSA using the RNeasy kit (Qiagen, Hilden, Germany). cDNA was synthesized using SuperScript II reverse transcriptase and Oligo (dT) primers (Invitrogen, Carlsbad, CA) in a 20 μl reaction according to the manufacturer's protocol. For the PCR reactions, Platinum SYBR Green qPCR SuperMix-UDG with ROX (Invitrogen, Carlsbad, CA) was used and reactions were assayed in triplicate using 1 μl of cDNA as a template in a 25 μl reaction. β-actin was used as the reference gene. Primers for TNFRSF10C were: 5'-TAC ACC AAC GCT TCC AAC-3' (forward) and 5'-AAG TGT AGC AGG TGC CCT-3' (reverse). TNFRSF10C PCR was performed with the following cycles: 95 ℃ for 10 minutes, 95 ℃ for 15 seconds, 63 ℃ for 32 seconds, and 72 ℃ for 30 seconds. A total of 40 cycles was performed and each reaction was run in duplicate. Negative controls were included in each experiment. Gene expression was calculated based on the yield of TNFRSF10C relative to β-actin.

    Results

    Apoptosis before and after drug treatment

    The percentage of apoptotic cells after treatment with 5-aza-dC and/or TSA was greater than that of the control in FCM and the TUNEL assay (Fig. 1). Higher levels of apoptosis were measured in CFPAC-1, PANC-1, and SW1990 cells treated with 5-aza-dC than with TSA. In contrast, the cell line BxPC-3 exhibited higher levels of apoptosis after TSA treatment (Fig. 1B).

    Methylation frequency of CGI in TNFRSF10C promoter

    The methylation frequency of the CGI in the TNFRSF10C promoter was assessed in PC cell lines using the COBRA assay. Methylation was absent in CFPAC-1 cells, 96.77±4.57% in PANC-1 cells, 68.84±8.71% in BxPC-3 cells, and 54.97±7.33% in SW1990 cells (Fig. 2A, B).

    At the same time, the DNAs of PANC-1 and CFPAC-1 were subjected to BGS. Ten CpG sites within the CGI of the TNFRSF10C promoter were detected and the percentages of methylation (PANC-1: 98%;CFPAC-1: 0%) were consistent with the results of the COBRA assay (Fig. 2C).

    Fig. 1. Evaluation of apoptosis. A: Flow cytometric analysis of apoptosis exhibited by PANC-1 cells after treatment with 5-aza-dC and/or TSA compared to control cells. B: The different apoptosis of PC cell lines treated with the combination of 5-aza-dC and TSA. BxPC-3 was more strongly affected by histone modification than changes in methylation. *, #, △: P<0.05, compared with control group. C-F: Representative images of apoptosis detected by TUNEL assay (original magnification ×400); C: untreated PANC-1 cells; D: negative control; E: PANC-1 cells treated with TSA; F: PANC-1 cells treated with 5-aza-dC. Blue staining indicates the absence of apoptosis and brown staining indicates the presence of apoptosis.

    Fig. 2. Methylation frequency of CGI in TNFRSF10C promoter. A: COBRA results from four PC cell lines (M: marker; B: BxPC-3; C: CFPAC-1; P: PANC-1; S: SW1990; SSSI: DNA treated with CpG methyltransferase). B: The methylation rate of TNFRSF10C was calculated from the level of the gray lane (Fig. 2A) according to the following formula: methylation rate (%)=(lane 2+lane 3)/(lane 1+lane 2+lane 3). C: BGS of PANC-1 (98%), CFPAC-1 (0%).

    Diversity of expression and changes in methylation before and after drug treatment

    Real-time RT-PCR further demonstrated that demethylation was accompanied by a 8.28-fold (5-azadC) and a 5.75-fold (TSA) increase in transcriptional expression of TNFRSF10C in PANC-1, and a 2.86-fold (5-aza-dC) and a 1.70-fold (TSA) increase in SW1990 (Fig. 3A). Unlike the other cell lines, TNFRSF10C was found to be more strongly expressed after TSA treatment (3.64-fold) than 5-aza-dC treatment (2.98-fold) in BxPC-3 cells, which was paralleled by the FCM results (Fig. 1B). These data suggest that silencing of the TNFRSF10C gene is achieved through promoter methylation.

    After 5-aza-dC or TSA treatment, BGS was used to re-assess the methylation status of TNFRSF10C in PANC-1 cells. The methylation rate in the untreated group was 98%, in the TSA group 70%, and in the 5-aza-dC group 40% (Fig. 3B).

    Fig. 3. Reduced methylation and reactivation of TNFRSF10C expression in PC cell lines after treatment with 5-aza-dC or TSA. A: Quantitative RT-PCR of TNFRSF10C mRNA after treatment of PC cell lines (CFPAC-1 is omitted) with 5-aza-dC or TSA. Untreated cells were used as control in each case. *, #: P<0.05, compared with the control group. B: BGS of the CGI of TNFRSF10C after treatment with 5-aza-dC or TSA in PANC-1 (untreated: 98%; TSA: 70%; 5-aza-dC: 40%).

    Discussion

    COBRA is a sensitive and quantitative assay to determine DNA methylation levels at specific gene loci in small amounts of genomic DNA. Restriction enzyme digestion is used to reveal methylation-dependent sequence differences in the PCR products of sodium bisulfitetreated DNA, and the results are verified by BGS. We evaluated the aberrant methylation frequency of TNFRSF10C in PC cell lines with the COBRA assay. Using a combined approach of pharmacologic inhibition of epigenetic modifications and gene expression assays, genes subject to epigenetic silencing in PC cell lines were identified. This strategy is also used to identify the targets of epigenetic silencing in other tumor types.[7,16-18]

    TNFRSF10C lacks both a cytoplasmic and a transmembrane domain, leaving it unable to participate in apoptotic signaling. Since a direct correlation between TNFRSF10C expression and TRAIL sensitivity has not been demonstrated,[19-21]the role of TNFRSF10C in PC is still unknown.

    In our study, hypermethylation of the TNFRSF10C promoter was found in all PC cell lines except CFPAC-1, but the mechanism is unclear. The results demonstrated a high prevalence of TNFRSF10C promoter CGI hyper-methylation in most PC cell lines. And the results of real-time RT-PCR showed low transcription in PC cells because of hypermethylation. After treatment with 5-aza-dC or TSA, we found that the methylation rate of the TNFRSF10C promoter CGI was cut from 98% to 40% (5-aza-dC) and to 70% (TSA) in PANC-1, and the transcriptional expression was enhanced by 8.28-fold (5-aza-dC) and 5.75-fold (TSA). Meanwhile, the apoptotic rate increased markedly. Thus gene silencing of TNFRSF10C is mostly due to aberrant hypermethylation, which plays an important role in the progression of PC. At the same time, 5-aza-dC and TSA have synergistic effects on demethylation to a certain extent, but the mechanism of demethylation by TSA is not clear. In the present study COBRA and BGS showed no methylation of the TNFRSF10C promoter in CFPAC-1 while apoptosis was still induced after 5-aza-dC and/or TSA treatment. The possible reason is that other anti-oncogenes were re-activated because there was no specific demethylation effect of 5-aza-dC. Both apoptosis and transcription of BxPC-3 treated with TSA were greater than that with 5-aza-dC, indicating that histone deacetylation mainly contributes to the progression in BxPC-3. On the contrary, hypermethylation plays a key role in progression in PANC-1 and SW1990. All the above suggest that different mechanisms result in tumorigenesis and progression in different PC cell lines.

    With the increasing incidence of morbidity and high mortality, PC responds poorly to conventional treatments. Evaluation of aberrant CGI methylation frequency in the TNFRSF10C promoter may be used as a biomarker of PC. The methylation status of the TNFRSF10C promoter should be investigated in clinical PC specimens before it is used as a new therapeutic approach for PC.

    Funding:The study was supported by a grant from the National Natural Science Foundation of China (30471691).

    Ethical approval:Not needed.

    Contributors:MY proposed the study. CHH and SYM carried out the study, and GWT, PQ and YJ collected and summarized the data. CHH wrote the first draft, which was corrected by MY and ZHL. All authors contributed to the intellectual context and approved the final version. MY is the guarantor.

    Competing interest:No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

    1 Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, et al. Cancer statistics, 2008. CA Cancer J Clin 2008;58:71-96.

    2 Ashkenazi A. Targeting death and decoy receptors of the tumour-necrosis factor superfamily. Nat Rev Cancer 2002;2: 420-430.

    3 Van Geelen CM, de Vries EG, de Jong S. Lessons from TRAIL-resistance mechanisms in colorectal cancer cells: paving the road to patient-tailored therapy. Drug Resist Updat 2004;7: 345-358.

    4 Tessema M, Yu YY, Stidley CA, Machida EO, Schuebel KE, Baylin SB, et al. Concomitant promoter methylation of multiple genes in lung adenocarcinomas from current, former and never smokers. Carcinogenesis 2009;30:1132-1138.

    5 Chaib H, MacDonald JW, Vessella RL, Washburn JG, Quinn JE, Odman A, et al. Haploinsufficiency and reduced expression of genes localized to the 8p chromosomal region in human prostate tumors. Genes Chromosomes Cancer 2003;37:306-313.

    6 Cheng Y, Kim JW, Liu W, Dunn TA, Luo J, Loza MJ, et al. Genetic and epigenetic inactivation of TNFRSF10C in human prostate cancer. Prostate 2009;69:327-335.

    7 Wu Y, Alvarez M, Slamon DJ, Koeffler P, Vadgama JV. Caspase 8 and maspin are downregulated in breast cancer cells due to CpG site promoter methylation. BMC Cancer 2010;10: 32.

    8 Shivapurkar N, Toyooka S, Toyooka KO, Reddy J, Miyajima K, Suzuki M, et al. Aberrant methylation of trail decoy receptor genes is frequent in multiple tumor types. Int J Cancer 2004; 109:786-792.

    2.科研成果轉化過程特征??蒲谐晒D化在不同的階段表現(xiàn)出不同的性質特點,有些是某一時點上的瞬間轉化,有些是一個比較緩慢的過程。不同階段不同形式的轉化,都需要借助一定的條件來完成,研究的任務是找到這些條件,促成轉化過程的完成。如表3所示:

    9 Adams J, Williams SV, Aveyard JS, Knowles MA. Loss of heterozygosity analysis and DNA copy number measurement on 8p in bladder cancer reveals two mechanisms of allelic loss. Cancer Res 2005;65:66-75.

    10 van Noesel MM, van Bezouw S, Salomons GS, Voute PA, Pieters R, Baylin SB, et al. Tumor-specific down-regulation of the tumor necrosis factor-related apoptosis-inducing ligand decoy receptors DcR1 and DcR2 is associated with dense promoter hypermethylation. Cancer Res 2002;62:2157-2161.

    11 Michalowski MB, de Fraipont F, Michelland S, Entz-Werle N, Grill J, Pasquier B, et al. Methylation of RASSF1A and TRAIL pathway-related genes is frequent in childhood intracranial ependymomas and benign choroid plexus papilloma. Cancer Genet Cytogenet 2006;166:74-81.

    12 Hornstein M, Hoffmann MJ, Alexa A, Yamanaka M, Müller M, Jung V, et al. Protein phosphatase and TRAIL receptor genes as new candidate tumor genes on chromosome 8p in prostate cancer. Cancer Genomics Proteomics 2008;5:123-136.

    13 Kim TY, Zhong S, Fields CR, Kim JH, Robertson KD. Epigenomic profiling reveals novel and frequent targets of aberrant DNA methylation-mediated silencing in malignant glioma. Cancer Res 2006;66:7490-7501.

    14 Warnecke PM, Stirzaker C, Song J, Grunau C, Melki JR, Clark SJ. Identification and resolution of artifacts in bisulfite sequencing. Methods 2002;27:101-107.

    15 Xiong Z, Laird PW. COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res 1997;25:2532-2534.

    17 Suzuki H, Gabrielson E, Chen W, Anbazhagan R, van Engeland M, Weijenberg MP, et al. A genomic screen for genes upregulated by demethylation and histone deacetylaseinhibition in human colorectal cancer. Nat Genet 2002;31:141-149.

    18 Lai JC, Wu JY, Cheng YW, Yeh KT, Wu TC, Chen CY, et al. O6-Methylguanine-DNA methyltransferase hypermethylation modulated by 17beta-estradiol in lung cancer cells. Anticancer Res 2009;29:2535-2540.

    19 Mahalingam D, Szegezdi E, Keane M, Jong S, Samali A. TRAIL receptor signalling and modulation: Are we on the right TRAIL? Cancer Treat Rev 2009;35:280-288.

    20 Tarragona J, Llecha N, Santacana M, Lopez S, Gatius S, Llobet D, et al. DcR1 expression in endometrial carcinomas. Virchows Arch 2010;456:39-44.

    21 Klugman SD, Gross SJ, Liang J, Livne K, Gross B, Khabele D, et al. Expression of Keratin 8 and TNF-Related Apoptosis-I Inducing Ligand (TRAIL) in Down Syndrome Placentas. Placenta 2008;29:382-384.

    Accepted after revision September 2, 2010

    Examine yourself every day. Correct the mistakes, if any, and guard against them, if not.

    – (the Song Dynasty) Zhu Xi

    May 20, 2010

    Author Affiliations: Department of General Surgery, First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, China (Cai HH, Sun YM, Miao Y, Gao WT, Peng Q, Yao J and Zhao HL); Department of General Surgery, Jiangsu Province Hospital of Traditional Chinese Medicine, Nanjing 210029, China (Cai HH)

    Yi Miao, MD, PhD, Department of General Surgery, First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, China (Tel: 86-25-83718836ext6508; Fax: 86-25-83781992; Email: miaoyi@njmu. edu.cn)

    ? 2011, Hepatobiliary Pancreat Dis Int. All rights reserved.

    猜你喜歡
    科研成果性質形式
    科研成果轉化
    水運工程(2022年7期)2022-07-29 08:36:12
    隨機變量的分布列性質的應用
    完全平方數(shù)的性質及其應用
    中國科研成果震撼全球
    九點圓的性質和應用
    微型演講:一種德育的新形式
    厲害了,我的性質
    搞定語法填空中的V—ing形式
    加強醫(yī)療科技自主創(chuàng)新和科研成果轉化
    審批復雜 科研成果落地難
    亚洲精品日韩av片在线观看| videossex国产| 久久久久久久久久成人| 日韩 亚洲 欧美在线| 国内揄拍国产精品人妻在线| 国产高清三级在线| 丝袜脚勾引网站| 国产成人精品婷婷| 久久女婷五月综合色啪小说| 午夜福利,免费看| 蜜桃久久精品国产亚洲av| 伦理电影免费视频| 久久久午夜欧美精品| 久久婷婷青草| 天堂俺去俺来也www色官网| 天美传媒精品一区二区| 国产熟女午夜一区二区三区 | 在线精品无人区一区二区三| 欧美日韩在线观看h| 男人狂女人下面高潮的视频| 热re99久久国产66热| 桃花免费在线播放| 在线观看免费日韩欧美大片 | 男女无遮挡免费网站观看| 国产精品伦人一区二区| 欧美另类一区| 日韩av在线免费看完整版不卡| 国产免费福利视频在线观看| 九草在线视频观看| 国产片特级美女逼逼视频| 久久国内精品自在自线图片| 亚洲婷婷狠狠爱综合网| av在线播放精品| 久久精品久久精品一区二区三区| 97在线视频观看| 日本wwww免费看| 夜夜看夜夜爽夜夜摸| h日本视频在线播放| 岛国毛片在线播放| 国产探花极品一区二区| 国产色爽女视频免费观看| 自线自在国产av| av在线app专区| 如何舔出高潮| 久久久久久久大尺度免费视频| 热re99久久精品国产66热6| 麻豆精品久久久久久蜜桃| 在现免费观看毛片| 精品国产国语对白av| 国产成人a∨麻豆精品| 国产精品欧美亚洲77777| 亚洲一级一片aⅴ在线观看| 日韩不卡一区二区三区视频在线| freevideosex欧美| 三级经典国产精品| 国产毛片在线视频| 人人妻人人看人人澡| 成人午夜精彩视频在线观看| 国产日韩欧美亚洲二区| 国产精品久久久久久久久免| 在线免费观看不下载黄p国产| 嫩草影院入口| 国产精品一区二区在线不卡| 各种免费的搞黄视频| 精品国产乱码久久久久久小说| 男人舔奶头视频| 夜夜看夜夜爽夜夜摸| 99视频精品全部免费 在线| 久久久久久久久久人人人人人人| 在线精品无人区一区二区三| 中国美白少妇内射xxxbb| 成人美女网站在线观看视频| 人妻系列 视频| 国产精品久久久久久精品电影小说| 91精品国产国语对白视频| 啦啦啦中文免费视频观看日本| 少妇被粗大的猛进出69影院 | av天堂中文字幕网| 夜夜骑夜夜射夜夜干| 国产精品嫩草影院av在线观看| 亚洲精品色激情综合| 欧美精品一区二区大全| 天堂中文最新版在线下载| 大香蕉久久网| 精品亚洲成国产av| 老熟女久久久| 国产深夜福利视频在线观看| 麻豆乱淫一区二区| 亚洲综合精品二区| 午夜91福利影院| 热re99久久国产66热| 大片电影免费在线观看免费| 亚洲欧美成人综合另类久久久| 最黄视频免费看| 欧美亚洲 丝袜 人妻 在线| 永久免费av网站大全| 久久久久久久久久人人人人人人| 一个人看视频在线观看www免费| 97在线人人人人妻| 极品教师在线视频| 亚洲美女搞黄在线观看| 亚洲av综合色区一区| 国产精品久久久久久久久免| 水蜜桃什么品种好| 9色porny在线观看| 久久综合国产亚洲精品| 18禁裸乳无遮挡动漫免费视频| 久久久久精品久久久久真实原创| 三级经典国产精品| 人妻制服诱惑在线中文字幕| 免费大片黄手机在线观看| 另类亚洲欧美激情| 国产伦理片在线播放av一区| 伊人久久精品亚洲午夜| 欧美人与善性xxx| 人人妻人人看人人澡| 国产淫语在线视频| 中文字幕人妻熟人妻熟丝袜美| 久久久久精品性色| 在线 av 中文字幕| 少妇猛男粗大的猛烈进出视频| 六月丁香七月| 一区二区三区四区激情视频| 久久97久久精品| 欧美日韩视频高清一区二区三区二| 男女边吃奶边做爰视频| 99久久精品热视频| 少妇被粗大猛烈的视频| 欧美日韩视频精品一区| 亚洲综合色惰| 日韩大片免费观看网站| 五月玫瑰六月丁香| 亚洲欧美成人综合另类久久久| 亚洲怡红院男人天堂| 自拍欧美九色日韩亚洲蝌蚪91 | 亚洲婷婷狠狠爱综合网| av女优亚洲男人天堂| 亚洲精品日本国产第一区| 久久人人爽人人片av| 国产69精品久久久久777片| 少妇 在线观看| 国产精品一二三区在线看| 99热全是精品| 99热6这里只有精品| 国产精品伦人一区二区| 极品少妇高潮喷水抽搐| 秋霞伦理黄片| 国产欧美亚洲国产| 精品熟女少妇av免费看| 成人毛片60女人毛片免费| 男人舔奶头视频| 免费播放大片免费观看视频在线观看| 最近最新中文字幕免费大全7| 国产男女内射视频| 日本av手机在线免费观看| 高清不卡的av网站| 夜夜骑夜夜射夜夜干| 人妻少妇偷人精品九色| 水蜜桃什么品种好| 亚洲精品456在线播放app| 亚洲第一av免费看| 亚洲性久久影院| 亚洲欧美日韩东京热| 欧美人与善性xxx| 国产成人freesex在线| 女人久久www免费人成看片| 色5月婷婷丁香| 黄色配什么色好看| 欧美激情极品国产一区二区三区 | 亚洲精品色激情综合| 午夜免费鲁丝| h视频一区二区三区| 又黄又爽又刺激的免费视频.| 精品人妻一区二区三区麻豆| 中国美白少妇内射xxxbb| 中文资源天堂在线| 久久毛片免费看一区二区三区| 成人午夜精彩视频在线观看| 麻豆成人av视频| .国产精品久久| 国产白丝娇喘喷水9色精品| 国产精品一区二区在线观看99| 黑丝袜美女国产一区| 91午夜精品亚洲一区二区三区| 久久国产乱子免费精品| 18禁裸乳无遮挡动漫免费视频| 国产精品一区二区在线不卡| 在线观看美女被高潮喷水网站| 一区二区三区免费毛片| 麻豆成人午夜福利视频| 美女cb高潮喷水在线观看| 久久精品国产亚洲网站| 国产精品久久久久久精品电影小说| 亚洲av综合色区一区| 久久久国产欧美日韩av| 久久狼人影院| 国产伦在线观看视频一区| 国产精品一区二区在线观看99| 美女脱内裤让男人舔精品视频| 男的添女的下面高潮视频| 国产一区二区三区av在线| 国产成人免费观看mmmm| 色哟哟·www| 91精品伊人久久大香线蕉| 一级毛片我不卡| 老司机影院成人| 国产在视频线精品| 我要看黄色一级片免费的| 一本久久精品| 高清不卡的av网站| 国产老妇伦熟女老妇高清| 超碰97精品在线观看| 亚洲精品国产av蜜桃| 九九爱精品视频在线观看| 日本91视频免费播放| 日韩精品免费视频一区二区三区 | 日日撸夜夜添| 夜夜骑夜夜射夜夜干| 国产一区有黄有色的免费视频| a级片在线免费高清观看视频| 久热这里只有精品99| 美女脱内裤让男人舔精品视频| 国内少妇人妻偷人精品xxx网站| 欧美精品国产亚洲| 亚洲四区av| 成人综合一区亚洲| 久久99一区二区三区| 欧美日韩精品成人综合77777| 少妇精品久久久久久久| 蜜桃久久精品国产亚洲av| 久久久久久久久久久丰满| 在线观看免费视频网站a站| 国产成人freesex在线| 天堂中文最新版在线下载| 欧美bdsm另类| 久久ye,这里只有精品| 日本欧美国产在线视频| 纯流量卡能插随身wifi吗| 亚洲国产毛片av蜜桃av| 国产一区二区三区综合在线观看 | 亚洲欧洲国产日韩| 日本猛色少妇xxxxx猛交久久| 免费观看无遮挡的男女| 丝袜脚勾引网站| 成年av动漫网址| 另类亚洲欧美激情| 国产探花极品一区二区| 欧美国产精品一级二级三级 | 成人国产av品久久久| 亚洲第一av免费看| 狠狠精品人妻久久久久久综合| 精品视频人人做人人爽| 欧美激情国产日韩精品一区| 精品国产露脸久久av麻豆| 国产一区二区三区av在线| xxx大片免费视频| 亚洲色图综合在线观看| 中文乱码字字幕精品一区二区三区| 精品99又大又爽又粗少妇毛片| 在线观看免费视频网站a站| 成人午夜精彩视频在线观看| 久久久精品免费免费高清| 亚洲av综合色区一区| 久久精品国产亚洲av天美| 男人添女人高潮全过程视频| 成年人免费黄色播放视频 | 一本色道久久久久久精品综合| 亚洲成人手机| 99久久精品国产国产毛片| 欧美日韩av久久| 99久久人妻综合| 色5月婷婷丁香| 大话2 男鬼变身卡| 国产精品福利在线免费观看| 亚洲自偷自拍三级| 精品少妇久久久久久888优播| 亚洲国产最新在线播放| 人妻系列 视频| 成年av动漫网址| 欧美变态另类bdsm刘玥| 人体艺术视频欧美日本| 69精品国产乱码久久久| 色婷婷av一区二区三区视频| 99九九线精品视频在线观看视频| a 毛片基地| 久久国产精品男人的天堂亚洲 | 亚洲综合色惰| 99九九线精品视频在线观看视频| 欧美日韩av久久| 久久精品熟女亚洲av麻豆精品| 成人亚洲精品一区在线观看| 看非洲黑人一级黄片| 亚洲精品国产色婷婷电影| 国语对白做爰xxxⅹ性视频网站| 欧美日韩亚洲高清精品| 精品久久久噜噜| 国产成人精品久久久久久| 精品久久久久久电影网| 大片免费播放器 马上看| 国产精品蜜桃在线观看| 尾随美女入室| 亚洲av中文av极速乱| 97在线视频观看| 亚洲国产精品成人久久小说| 亚洲精品中文字幕在线视频 | 免费黄网站久久成人精品| 欧美一级a爱片免费观看看| 在线观看人妻少妇| 在线观看免费视频网站a站| 夜夜爽夜夜爽视频| 欧美三级亚洲精品| 亚洲怡红院男人天堂| 综合色丁香网| 久久精品久久久久久久性| 国产精品99久久久久久久久| freevideosex欧美| 国产精品偷伦视频观看了| 夫妻午夜视频| 日韩 亚洲 欧美在线| 国产欧美日韩精品一区二区| 夫妻性生交免费视频一级片| 观看av在线不卡| 91久久精品国产一区二区成人| 亚洲av综合色区一区| 麻豆成人av视频| 亚洲av中文av极速乱| 国产伦精品一区二区三区视频9| 男女国产视频网站| 青春草国产在线视频| 97超碰精品成人国产| 男女边摸边吃奶| 2018国产大陆天天弄谢| 热99国产精品久久久久久7| 成人美女网站在线观看视频| 一级毛片黄色毛片免费观看视频| 亚洲综合色惰| 夜夜骑夜夜射夜夜干| 少妇精品久久久久久久| 大香蕉久久网| 亚洲激情五月婷婷啪啪| 男女国产视频网站| 久久国内精品自在自线图片| 极品教师在线视频| 久久精品国产亚洲av涩爱| 少妇被粗大猛烈的视频| 一级二级三级毛片免费看| 狂野欧美激情性bbbbbb| av在线app专区| 在线天堂最新版资源| 精品国产一区二区久久| 国产黄片视频在线免费观看| 国产一级毛片在线| 九草在线视频观看| 精品国产一区二区久久| 免费观看无遮挡的男女| 久久精品国产a三级三级三级| 久久女婷五月综合色啪小说| 国产精品偷伦视频观看了| 一级毛片久久久久久久久女| 亚洲精品中文字幕在线视频 | 亚洲欧美一区二区三区黑人 | www.色视频.com| 久久综合国产亚洲精品| 一级黄片播放器| 色5月婷婷丁香| 汤姆久久久久久久影院中文字幕| 9色porny在线观看| 国产精品伦人一区二区| 精品酒店卫生间| 精品国产一区二区久久| 成人无遮挡网站| 久久精品国产鲁丝片午夜精品| 在线天堂最新版资源| 亚洲自偷自拍三级| 狂野欧美激情性bbbbbb| 熟女av电影| 亚洲一区二区三区欧美精品| 中国国产av一级| 成人特级av手机在线观看| 插阴视频在线观看视频| av在线老鸭窝| 寂寞人妻少妇视频99o| 中文精品一卡2卡3卡4更新| 久久精品夜色国产| 欧美人与善性xxx| 亚洲国产欧美在线一区| 久久99热6这里只有精品| 麻豆成人av视频| 亚洲伊人久久精品综合| 一本久久精品| 成人美女网站在线观看视频| 美女大奶头黄色视频| 亚洲精品国产色婷婷电影| 丰满饥渴人妻一区二区三| 精品人妻熟女毛片av久久网站| 69精品国产乱码久久久| 欧美日韩国产mv在线观看视频| 亚洲国产精品国产精品| 三级国产精品欧美在线观看| 亚洲欧洲精品一区二区精品久久久 | 日韩不卡一区二区三区视频在线| 嫩草影院新地址| 亚洲国产精品国产精品| 人妻夜夜爽99麻豆av| 国产高清国产精品国产三级| 超碰97精品在线观看| 久久久久久久久久久免费av| 99热网站在线观看| 老司机影院成人| 欧美老熟妇乱子伦牲交| 亚洲国产av新网站| 亚洲四区av| 亚洲欧美成人精品一区二区| 又大又黄又爽视频免费| 婷婷色综合www| 日韩电影二区| 在线观看免费日韩欧美大片 | a级毛片免费高清观看在线播放| 亚洲国产成人一精品久久久| 看免费成人av毛片| 亚洲国产精品专区欧美| a 毛片基地| 99re6热这里在线精品视频| 亚洲熟女精品中文字幕| 水蜜桃什么品种好| 精品国产一区二区三区久久久樱花| 蜜臀久久99精品久久宅男| 99视频精品全部免费 在线| 草草在线视频免费看| 51国产日韩欧美| 国产高清不卡午夜福利| 久久ye,这里只有精品| 一级爰片在线观看| 日韩精品有码人妻一区| 婷婷色麻豆天堂久久| 777米奇影视久久| 国产精品久久久久久久久免| 美女脱内裤让男人舔精品视频| 伦理电影免费视频| 国产日韩欧美亚洲二区| 亚洲av国产av综合av卡| 国产成人a∨麻豆精品| 国产欧美日韩精品一区二区| 国产精品成人在线| 精品久久久精品久久久| 亚洲无线观看免费| 一本—道久久a久久精品蜜桃钙片| 免费在线观看成人毛片| 成人亚洲精品一区在线观看| 国产精品三级大全| 丝瓜视频免费看黄片| 亚洲激情五月婷婷啪啪| 亚洲婷婷狠狠爱综合网| 久久精品熟女亚洲av麻豆精品| 少妇被粗大猛烈的视频| 亚洲国产精品成人久久小说| 亚洲无线观看免费| 久久久久视频综合| 秋霞在线观看毛片| 最近中文字幕2019免费版| 亚洲欧洲精品一区二区精品久久久 | 秋霞在线观看毛片| 2021少妇久久久久久久久久久| 尾随美女入室| 日韩中文字幕视频在线看片| 亚洲成人一二三区av| 两个人的视频大全免费| 黄色毛片三级朝国网站 | 七月丁香在线播放| 麻豆乱淫一区二区| 久久人妻熟女aⅴ| 久久97久久精品| 80岁老熟妇乱子伦牲交| 女人精品久久久久毛片| 欧美 日韩 精品 国产| 久久热精品热| 亚洲精品国产av成人精品| 黄色配什么色好看| 黑人猛操日本美女一级片| 伊人久久国产一区二区| av在线观看视频网站免费| 美女cb高潮喷水在线观看| 午夜福利,免费看| 国产精品三级大全| 老女人水多毛片| 精华霜和精华液先用哪个| 精品一区二区三区视频在线| 大香蕉久久网| 久久午夜综合久久蜜桃| 日韩视频在线欧美| 美女国产视频在线观看| 国产69精品久久久久777片| 久久99一区二区三区| 精品卡一卡二卡四卡免费| 99国产精品免费福利视频| 激情五月婷婷亚洲| 伊人久久国产一区二区| 最黄视频免费看| 国产日韩欧美视频二区| 制服丝袜香蕉在线| 精品国产国语对白av| 伊人久久精品亚洲午夜| 国产综合精华液| 亚洲国产精品999| 我要看日韩黄色一级片| 少妇的逼水好多| 日韩大片免费观看网站| 如何舔出高潮| 日日啪夜夜撸| 亚洲国产色片| 亚洲电影在线观看av| 亚洲成人手机| 欧美激情极品国产一区二区三区 | 久久毛片免费看一区二区三区| 大码成人一级视频| 精品亚洲乱码少妇综合久久| 国产真实伦视频高清在线观看| 国产美女午夜福利| 纵有疾风起免费观看全集完整版| 插阴视频在线观看视频| h日本视频在线播放| 免费大片18禁| 免费看不卡的av| 少妇的逼好多水| 丝袜喷水一区| 亚洲怡红院男人天堂| 日本黄色片子视频| 欧美亚洲 丝袜 人妻 在线| 国产黄色视频一区二区在线观看| 精品熟女少妇av免费看| 国模一区二区三区四区视频| 久久久久国产网址| 在线观看美女被高潮喷水网站| 另类亚洲欧美激情| 国产男女内射视频| 黄色欧美视频在线观看| 国产精品国产三级国产av玫瑰| 三上悠亚av全集在线观看 | 简卡轻食公司| 亚洲欧美一区二区三区黑人 | 热99国产精品久久久久久7| av有码第一页| 国精品久久久久久国模美| 国产毛片在线视频| 搡老乐熟女国产| 久久久欧美国产精品| 国产男女内射视频| 国产淫语在线视频| 国产亚洲欧美精品永久| 丝袜喷水一区| 99久久中文字幕三级久久日本| 亚洲不卡免费看| 日韩制服骚丝袜av| 黑人巨大精品欧美一区二区蜜桃 | 国产精品伦人一区二区| 少妇人妻一区二区三区视频| 成年av动漫网址| 亚洲怡红院男人天堂| 熟妇人妻不卡中文字幕| 美女主播在线视频| 超碰97精品在线观看| 免费人妻精品一区二区三区视频| 韩国av在线不卡| 91久久精品电影网| 日韩亚洲欧美综合| 边亲边吃奶的免费视频| 亚洲精品日本国产第一区| 插逼视频在线观看| 在线观看免费高清a一片| 日韩熟女老妇一区二区性免费视频| 一二三四中文在线观看免费高清| 精品酒店卫生间| 嫩草影院入口| 大香蕉久久网| 毛片一级片免费看久久久久| 亚洲经典国产精华液单| 国产免费一区二区三区四区乱码| 国产精品.久久久| 久久韩国三级中文字幕| 黄色日韩在线| 午夜91福利影院| 亚洲电影在线观看av| 国产精品久久久久成人av| 国产成人精品久久久久久| 午夜老司机福利剧场| 国产av码专区亚洲av| 2021少妇久久久久久久久久久| 全区人妻精品视频| 五月天丁香电影| 久久久亚洲精品成人影院| 久久精品夜色国产| 亚洲精品久久久久久婷婷小说| 国产视频内射| 一区二区三区精品91| 另类精品久久| 国产欧美日韩综合在线一区二区 | av在线老鸭窝| 午夜激情久久久久久久| 亚洲丝袜综合中文字幕| 黑人猛操日本美女一级片| kizo精华| 免费大片18禁| 少妇高潮的动态图| 黄色毛片三级朝国网站 | 校园人妻丝袜中文字幕| 九草在线视频观看| 亚洲婷婷狠狠爱综合网| 国产有黄有色有爽视频| 国产亚洲精品久久久com| 天天操日日干夜夜撸| 国产黄色视频一区二区在线观看| 亚洲精品日韩在线中文字幕| 韩国高清视频一区二区三区| 日韩中字成人| 国产色爽女视频免费观看|