• <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 ?

    Value of long non-coding RNA Rpph1 in esophageal cancer and its effect on cancer cell sensitivity to radiotherapy

    2020-05-07 08:55:22ZhenYangLiHuiFenLiYingYingZhangXueLanZhangBingWangJiangTingLiu
    World Journal of Gastroenterology 2020年15期

    Zhen-Yang Li, Hui-Fen Li, Ying-Ying Zhang, Xue-Lan Zhang, Bing Wang, Jiang-Ting Liu

    Abstract BACKGROUND Esophageal cancer is a common digestive tract tumor that is generally treated with radiotherapy. Poor responses to radiotherapy in most patients generally result in local radiotherapy failure, so it is essential to find new radiosensitizers that can enhance the response of cancer cells to radiotherapy and improve the survival of esophageal cancer patients with radiation resistance. The long noncoding RNA (lncRNA) Rpph1 is highly expressed in human gastric cancer tissues, and represses breast cancer cell proliferation and tumorigenesis.However, the expression of lncRNA Rpph1 in esophageal cancer and its relationship with radio-sensitivity has not been studied.AIM To explore the value of lncRNA Rpph1 in esophageal cancer and its effect on cancer cell sensitivity to radiotherapy.METHODS Eighty-three patients with esophageal cancer admitted to Qilu Hospital of Shandong University and 90 healthy participants who received physical examinations were collected as research participants. The expression of Rpph1 was determined by qRT-PCR. siRNA-NC and siRNA-Rpph1 were transfected into esophageal cancer cell lines, and cells without transfection were designated as the blank control group. Cell survival was tested by colony formation assays,and the levels of proteins related to apoptosis and epithelial-mesenchymal transitions were determined by Western blot assays. Cell proliferation was assessed by MTT assays, cell apoptosis by flow cytometry, and cell migration by wound-healing assays. Changes in cell cycle distribution were monitored.RESULTS Rpph1 was highly expressed in esophageal carcinoma, making it a promising marker for the diagnosis of esophageal cancer. Rpph1 could also be used to distinguish different short-term responses, T stages, N stages, and clinical stages of esophageal cancer patients. The results of 3-year overall survival favored patients with lower Rpph1 expression over patients with higher Rpph1 expression (P < 0.05). In vitro and in vivo experiments showed that silencing Rpph1 expression led to higher sensitivity of esophageal cancer cells to radiotherapy, stronger apoptosis in esophageal cancer cells induced by radiotherapy, higher expression of Bax and caspase-3, and lower expression of Bcl-2 (Bax, caspase-3, and Bcl-2 are apoptosis-related proteins). Additionally,silencing Rpph1 attenuated radiation-induced G2/M phase arrest, and significantly inhibited the expression of proteins involved in cell proliferation,migration, and epithelial-mesenchymal transition regulation in esophageal cancer cells.CONCLUSION Rpph1 is highly expressed in esophageal cancer. Silencing Rpph1 expression can promote cell apoptosis, inhibit cell proliferation and migration, and increase radio-sensitivity.

    Key words: Long non-coding RNA Rpph1; Esophageal cancer; Cell sensitivity to radiotherapy; Apoptosis; Cell cycle; Epithelial-mesenchymal transition

    INTRODUCTION

    Esophageal cancer is the eighth most prevalent cancer worldwide, as well as one of the main causes of death due to cancer in human[1,2]. The annual mortality of esophageal cancer is approximately 100/100000 people, with the highest incidence in Asia, northern France, eastern and southern Africa[3]. Esophageal squamous cell carcinoma and esophageal adenocarcinoma are the two major histological types of esophageal cancer[4]. Due to the lack of specific early symptoms, obvious signs of onset, and early diagnostic markers, most patients with esophageal cancer are already at advanced cancer stages at the time of treatment, demonstrating poor treatment response following surgical resection[5]. Radiotherapy is crucial for the treatment of patients not qualified for operations and those with locally advanced esophageal cancer[6]. Local radiotherapy failure may occur due to radiotherapy resistance or tumor heterogeneity. Persistent or recurrent disease is reported in 40%-60% of esophageal cancer patients receiving radiotherapy, and the prognosis of esophageal cancer is awful[7,8]. Therefore, the search for new radiosensitizers that can enhance radio-sensitivity in tumor cells and improve the survival of esophageal cancer patients with radiation-resistance is clinically urgent[9,10].

    The identification of molecular markers may improve the staging of esophageal cancer patients and develop new targeted therapies[11]. Long non-coding RNAs(lncRNAs), over 200 bp in length, have become the focus of public attention[12].lncRNAs can interact with multiple RNA molecules or proteins both transcriptionally and post-transcriptionally to control the expression of target genes[13-15]. Related studies reported that lncRNA is crucial for dose compensation, epigenetic regulation,and regulation of cell cycle and cell differentiation[16,17]. The lncRNA Rpph1 is abnormally expressed in the neocortical tissue of patients with gastric cancer[18]and epilepsy[19]. A study revealed that lncRNA Rpph1 could inhibit breast cancer cell proliferation and tumor occurrence[20]. The study by Chenget al[11]showed that XRCC 3 could increase the radio-sensitivity of esophageal cancer, so it is a latent target for treating esophageal cancer. So far, no study has been reported on lncRNA Rpph1 expression in esophageal cancer and its effect on the sensitivity to radiotherapy.

    This study measured lncRNA Rpph1 expression in esophageal cancer, aiming to investigate the clinical value of lncRNA Rpph1 in esophageal cancer and its effect on cancer cell sensitivity to radiotherapy.

    MATERIALS AND METHODS

    General information

    Eighty-three patients with esophageal cancer admitted to Qilu Hospital of Shandong University were enrolled, including 48 males and 35 females whose average age was(63.28 ± 8.83) years. Meanwhile, 90 healthy people who underwent physical examinations in Qilu Hospital of Shandong University during the same period were also enrolled, including 47 males and females whose average age was (62.65 ± 8.31)years. The general information of the two groups was not markedly different. With detailed descriptions of the experimental content, this study obtained approval from the Ethics Committee of Qilu Hospital of Shandong University. All participants signed a written informed consent. Inclusion criteria: Patients diagnosed with esophageal cancer by cytological and histopathological examination; patients with complete clinical data and good compliance; patients without mental illness; patients with an expected survival of longer than 1 mo; patients with no other malignant tumors. Exclusion criteria: Those with severe organic failure; those undergoing other anti-tumor treatments within 1 mo prior to the treatment; those with autoimmune system defects.

    All patients received a concurrent radiotherapy regimen with a total dose of 60-70 Gy, 1.8-2.0 Gy each time, completed within 6-7 wk. All patients finished the complete course of treatment. Regular reexamination and follow-up were conducted. The follow-up was performed every 3 mo for 3 years.

    Experimental materials

    RPMI-1640 medium and fetal bovine serum were provided by GIBCO, Inc. (item numbers: 31800002, 10099141). TRIzol reagent was from Invitrogen (Carlsbad, CA,United States), 0.25% trypsin from GIBCO, PBS buffer from Nanjing SenBeiJia Biological Technology Co., Ltd. Dimethyl sulfoxide (DMSO) was provided by Sigma,PVDF membrane by Millipore, DR5000 UV-Vis spectrophotometer by BioRad(Hercules, CA, United States), real-time PCR instrument by ABI (item number: 7500;Foster City, CA, United States). SYBR Premix ExTaqTMwas manufactured by Takara(Shiga, Japan). RIPA buffer was from Shanghai Haling Biological Technology Co. Ltd.(Shanghai, China). BCA Protein Assay Kit was provided by Shanghai Yubo Biological Technology Co. Ltd.. Flow cytometry was from Beckman (Brea, CA, United States),Annexin V-FITC Apoptosis Kit from BestBio. Primary antibodies including casepase-3, Bax, Bcl-2, and β-action were provided by Santa Cruz Biotechnology (Dallas, TX,United States). Vimentin, E-cadherin, N-cadherin, and horseradish peroxidase (HRP)-labeled goat anti-mouse secondary antibody were manufactured by Abcam(Cambridge, United Kingdom). MTT kit was provided by Beijing Baiao Laibo Technology Co. Ltd. ECL luminescence kit and MultiskanTMGO full-wavelength microplate reader by Thermo Fisher Scientific China Co. Ltd. (Shanghai, China).

    Sample collection

    We collected cancer tissues and adjacent tissues from esophageal cancer patients and stored them in liquid nitrogen. For each patient and healthy participant, 5 mL of fasting elbow vein blood was taken and centrifuged at 3000 ×gfor 10 min, and then the serum was collected.

    Cell culture

    Esophageal cancer TE-1 and Kyse150 cell lines were provided by iCell Bioscience, Inc,Shanghai (item numbers: HDCL-040, HDCL-050). TE-1 and Kyse150 cells were cultured in RPMI1640 medium comprised of 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. After cell culture at 37 °C in an incubator with 5% CO2and saturated humidity, cell passage was performed and cells were cryopreserved. Cells in logarithmic growth phase were harvested.

    Cell transfection

    Grouping of transfection: Blank control group (not transfected), empty vector negative control group (siRNA-NC), Rpph1-silenced group (siRNA-Rpph1), empty vector combined with radiotherapy group (siRNA-NC + IR), Rpph1-silenced combined with radiotherapy group (siRNA-Rpph1 + IR). When the adherent growth of esophageal cancer cells from the TE-1 and Kyse150 lines reached 80%-90%, the transfection was carried out according to the manual of the LipofectamineTM2000 transfection kit (Invitrogen). After 6 h of transfection, cell culture was performed in a new medium containing 10% fetal bovine serum. Cell transfection efficiency was measured by qRT-PCR. After 24 h of transfection, X-ray irradiation treatment was performed before subsequent experimentation.

    qRT-PCR detection

    TRIzol extraction kit was used to extract total RNA from serum, tissues, and cells. The purity, concentration, and integrity of total RNA were measured by UV spectrophotometer and agarose gel electrophoresis. The RNA was reverse transcribed into cDNA based on the instructions from the reverse transcription kit and was then stored at -20 °C. PCR was conducted with the SYBR Premix Ex TaqTMkit on a real-time PCR instrument, with GAPDH as the internal reference. Forward primer of Rpph1: 5'-CAGACTGGGCAGGAGAAGCC -3', reverse primer of Rpph1: 5'-TCACCTCAGCCATTGAACTCG-3'.Forward primer of GAPDH: 5'-AAGGGTGGAGCCAAAAGGG-3', reverse primer of GAPDH: 5' -TGGGGGT AGGAACACGGAA-3'. PCR amplification conditions: 40 cycles of 95 °C for 30 s, 95 °C for 5 s, 60 °C for 30 s. The experiment was repeated 3 times to obtain the final data,which were calculated using 2-ΔCT.

    Colony formation assay

    The esophageal cancer TE-1 and Kyse150 cells in the logarithmic growth phase were digested with trypsin and diluted to a suitable concentration. Cell counting was conducted under a microscope. There were six exposure groups at different doses (0,2, 4, 6, 8 Gy, respectively). Cell culture was conducted in an incubator at 37 °C, with 5% CO2and 2 mL of culture solution. After 24 h of adherence, cells received irradiation at various doses. Cells were kept in the incubator after the end of irradiation. When macroscopic clones appeared, cell culture was stopped and PBS washing was conducted twice, followed by fixation with formaldehyde and staining with crystal violet. The number of clones with 50 or more cell was counted under a microscope and the colony formation rate was calculated. Calculation formula:Plating efficiency (PE) = number of clones per well in the blank control group/number of cells inoculated per well; surviving fraction (SF) = number of clones in an experimental group/(number of cells inoculated × PE). The experiment was done in triplicate to obtain the mean value and a colony formation curve was generated.

    Western blot detection

    RIPA lysis buffer was used to extract the total protein that was then separated by 10%SDS-PAGE. The protein density was determined by BCA method. The separated protein was transferred to a polyvinylidene fluoride membrane, which was performed at constant voltage (100 V) for 100 min and blocked at 37 °C for 60 min.The membrane was then blocked in 5% skim milk, followed by an immune reaction:the membrane was incubated with primary antibody (1:1000) overnight at 4 °C and then incubated with secondary antibody (1:1000) at 37 °C for 1 h. The whole system was rinsed three times with TBST, 5 min each time. After the completion of the immune reaction, color development and fixation were performed using ECL luminescence reagent. Images were acquired using the Quantity One infrared imaging system. The relative expression level of protein = band gray value/internal reference gray value.

    Apoptosis experiment

    After digestion with trypsin, two round of PBS washing followed. After being mixed with 500 μL binding buffer, cells were resuspended and transferred to a flow tube,and then the system was added to 5 μL Annexin V-FITC and 5 μL PI and incubated at room temperature for 5 min away from light. Cell apoptosis was measured on the Kurt CytoFLEX LX flow cytometry system (Beckman). The procedures were conducted in triplicate to obtain the average value.

    Cell cycle

    Cells were seeded in six-well plates and then exposed to irradiation at 0 Gy or 6 Gy according to the grouping of siRNA-NC, siRNA-Rpph1, siRNA-NC + IR, and siRNARpph1 + IR. After 24 h of cell culture, PBS washing was performed twice and cells were mixed with 200 μL PBS to obtain a cell suspension which was fixed in 70%ethanol at 4 °C for 2 h. After centrifugation at 1000 r/min for 5 min, cells were washed twice with PBS and mixed with PI stain and then incubated for 30 min away from light. Finally, the changes in esophageal cancer cell cycle in each transfection group were determined by flow cytometry and Single Histogram Statistics.

    MTT assay for cell proliferation

    Logarithmic growth cells were harvested and trypsinized before centrifugation. Cells were then inoculated into 96-well plates at a concentration of 4000 cells/well. Cells were cultured at 37 °C under 5% CO2for 24, 48, 72, and 96 h. The upper original medium was discarded, and 20 μL MTT solution and 150 μL dimethyl sulfoxide were added into each well. The plate was shaken for 5-10 min until all purple crystals disappeared. The optical density (OD) value of each well was measured at 450 nm by a microplate reader.

    Wound-healing assay for cell migration

    After cell counting, logarithmic growth phase cells were inoculated in plates and cultured in a medium with 2.5% fetal bovine serum. A scratch was made on cells with a sterilized 100 μL disposable pipette tip perpendicular to the horizontal plane and then cells were carefully washed three times with PBS before being transferred to serum-free medium. The scratches of each group were observed under a microscope to evaluate the cell migration at 0 h and 24 h after dissection.

    Determine of expression of epithelial-mesenchymal transition-related proteins

    The expression of epithelial-mesenchymal transition (EMT)-related proteins:cytoskeletal protein (Vimentin), epithelial cadherin (E-cadherin), and neuro-cadherin(N-cadherin) levels were determined by Western blot assay following the same steps as described above.

    Statistical processing

    Statistical analysis was performed sing SPSS19.0 (Asia Analytics Formerly SPSS China). The measurement data were expressed with the mean ± SD and compared between any two groups using independent samplet-tests, and between multiple groups using one-way analysis of variance. LSD-ttest was used as the post hoc test.The diagnostic value was assessed by the receiver operating characteristic (referred to as ROC) curve. Correlation analysis was performed using the Pearson correlation coefficient. The survival of patients was displayed in a Kaplan-Meier survival curve and compared by Log-rank test. A statistical difference was determined forP< 0.05.

    RESULTS

    Expression of lncRNA Rpph1 in esophageal cancer and its clinical value

    Esophageal cancer tissues had higher lncRNA Rpph1 expression than tumor-adjacent tissues (P< 0.001). LncRNA Rpph1 expression was higher in the serum of esophageal cancer patients than in the serum of healthy participants (P< 0.001). Pearson analysis revealed a positive correlation between lncRNA Rpph1 expression in esophageal cancer tissues and in the serum of esophageal cancer patients (r= 0.6818,P< 0.001).ROC curves demonstrated that serum lncRNA Rpph1 for diagnosing esophageal cancer had an area under the curve (AUC) value of 0.9248, a sensitivity of 87.95%, and a specificity of 86.67%. More details are shown in Figure 1. The follow-up lasted for 3 years and revealed that 3 years of survival was achieved in 35 patients (42.17%). With the median value of serum lncRNA Rpph1 expression (2.060) as the division line,patients with a lncRNA Rpph1 expression higher than 2.060 were assigned into the high expression group (n= 41), and those with an expression equal to or lower than 2.060 were assigned into the low expression group (n= 42). The results of 3-year overall survival favored the low expression group over the high expression group(51.22%vs33.33%,P= 0.01).

    Relationship between lncRNA Rpph1 expression and clinicopathological features of esophageal cancer

    As displayed in Table 1, the expression of lncRNA Rpph1 varied in patients with different short-term responses, T stages, N stages, and other clinical stages (P< 0.05).ROC curves were employed to analyze the diagnostic value of lncRNA Rpph1 for different pathological parameters of esophageal cancer and revealed that lncRNA Rpph1 could be used to distinguish different short-term responses, T stages, N stages,and clinical stages. More details are shown in Figure 2 and Tables 1 and 2.

    Figure 1 Expression of long non-coding RNA Rpph1 in esophageal cancer and its clinical value. A: Expression of long non-coding RNA (lncRNA) Rpph1 in esophageal cancer tissues and adjacent tissues; B: Serum expression of lncRNA Rpph1 in esophageal cancer patients and healthy participants; C: Correlation between serum lncRNA Rpph1 expression and lncRNA Rpph1 expression in tissue; D: ROC analysis of the diagnostic value of serum lncRNA Rpph1 for patients with esophageal cancer; E: Survival curve analysis of patients with esophageal cancer; F: Survival curves of the Rpph1 high expression group and low expression group.

    Effect of lncRNA Rpph on cancer cell sensitivity to radiotherapy

    The blank control group, siRNA-NC, and siRNA-Rpph1 were designed according to the experimental requirements. The expression of Rpph1 in TE-1 and Kyse150 cells was detected by qRT-PCR 48 h after siRNA was transfected into TE-1 and Kyse150 cells. Comparison between the blank control group and the siRNA-NC group in Rpph1 expression showed no statistical difference, while the siRNA-Rpph1 group had significantly lower Rpph1 expression than the other two groups (P< 0.05). The colony formation assay demonstrated that the expression of siRNA-Rpph1 did not affect the colony sizes of TE-1 and Kyse150 cells. The surviving fractions of TE-1 and Kyse150 cells in the three groups before radiotherapy were not markedly different. The cell surviving fraction in each group decreased with increasing radiotherapy doses, with a greater decrease in the siRNA-Rpph1 group. The siRNA-Rpph1 group had a markedly smaller cell surviving fraction than the blank control group and the siRNANC group after radiotherapy (P< 0.05) (Figure 3).

    Effect of lncRNA Rpph1 on radiation-induced apoptosis and apoptosis proteins

    Esophageal cancer cell apoptosis in the blank control group, siRNA-NC group, and siRNA-Rpph1 group under different doses of radiotherapy were tested. Cell apoptosis rates of the three groups were not dramatically different before radiotherapy. After TE-1 and Kyse150 cells were exposed to irradiation at 4 Gy and 6 Gy, the siRNA-Rpph1 group had remarkably higher apoptosis rates than the blank control group and the siRNA-NC group (P< 0.001). Western Blot detection was employed to quantify the expression of apoptosis-related proteins in esophageal cancer cells before and after radiotherapy. As displayed in Figure 4, after cell exposure to 6 Gy irradiation, the siRNA-Rpph1 + IR group and the siRNA-NC group had ramped-up Bax and caspase-3 expression and a reduction in Bcl-2 expression.Cells in the siRNA-Rpph1 + IR group had remarkably higher expression of Bax and caspase-3 proteins, and lower expression of Bcl-2 proteins than cells in the siRNA-NC group.

    Effect of lncRNA Rpph1 on radiation-induced cell cycle

    Figure 5 shows that radiotherapy resulted in more G2/M phase cells. The siRNARpph1 + IR group (6 Gy) had markedly fewer G2/M phase cells than the siRNA-NC +IR group (P< 0.05), implying that downregulation of Rpph1 expression can effectively alleviate radiation-induced G2/M phase arrest, thereby enhancing the sensitivity of the esophageal cancer cell lines TE-1 and Kyse150 to radiotherapy.

    Table 1 Relationship between long non-coding RNA Rpph1 expression and clinicopathological features of esophageal cancer

    Effect of lncRNA Rpph1 on radiation-induced cell proliferation, migration, and EMT

    The OD values of TE-1 and Kyse150 cells in the three groups (siRNA-Rpph1, siRNANC, and blank control group) were measured by MTT assay at 24, 48, 72 and 96 h after cell culture. Comparison between the three groups in the OD value at 24 h showed no dramatic differences. The OD values of each group underwent a gradual ramp-up. The siRNA-Rpph1 group had slightly lower OD values than the other two groups at 48 h. No great differences in OD values were observed between the blank control group and the siRNA-NC group at each time point, while the OD values at 72 and 96 h were much lower in the siRNA-Rpph1 group than in the other two groups (P< 0.05). All three groups were exposed to different doses of irradiation (0, 2, 4, 6, and 8 Gy). The cell proliferation was weaker in the siRNA-Rpph1 group than in the blank control group and siRNA-NC group after each dose of irradiation (P< 0.05), while the blank control group and the siRNA-NC group were not different in cell proliferation rate. Cell migration was significantly weaker in the siRNA-Rpph1 group than in the siRNA-NC group (P< 0.05). After exposure to irradiation at 6 Gy, the cell migration ability of TE-1 and Kyse150 cells was poorer in the siRNA-Rpph1 group than in the siRNA-NC group (P< 0.05). Expression of EMT-related proteins: Cytoskeletal protein(Vimentin), epithelial cadherin (E-cadherin), and neuro-cadherin (N-cadherin)expression levels were determined. The siRNA-Rpph1 group had markedly lower expression of Vimentin and N-cadherin, and higher expression of E-cadherin than the siRNA-NC group (P< 0.05). After radiotherapy, the expression of Vimentin and Ncadherin in the siRNA-Rpph1 group significantly decreased, while showing much higher E-cadherin expression than the siRNA-NC group (P< 0.05) (Figure 6).

    DISCUSSION

    As one of the most prevalent malignant tumors, esophageal cancer has an incidence that increases with age[21]. Postoperative radiotherapy is an effective method to improve survival rates and reduce recurrence rates[22]. Surgery and radiotherapy are effective in esophageal cancer patients, but the 5-year survival rate is poor[21].LncRNA, involved in many complex regulatory networks, has become a research hotspot in recent years. Rpph1 is known as the subunit of RNase P RNA thatregulates tRNA maturation in all life domains[23], and it often works as an internal reference for RNA quantification[24-26]. A previous study suggested that Rpph1 may play a part in disease progression in animals and humans[27].

    Table 2 Diagnostic value of long non-coding RNA Rpph1 for different pathological parameters

    One related study has reported the upregulation of Rpph1 in human gastric cancer tissues[18]. Thus far, however, no studies on Rpph1 in esophageal cancer have been published. This study revealed high Rpph1 expression in esophageal cancer tissues and serum, implying the possibility that Rpph1 has an effect on the occurrence and development of esophageal cancer. Due to its accessibility and the limited invasiveness required for its collection, serum samples for the diagnosis of esophageal cancer are currently popular. The study by Wanget al[28]found that HOTAIR, a lncRNA transcribed from the HOXC locus, could be used as a novel diagnostic marker for esophageal squamous cell carcinoma by detecting its serum expression.This study reveals that serum lncRNA Rpph1 is associated with short-term responses,T stages, N stages, and clinical stages in patients with esophageal cancer, and can help diagnose esophageal cancer. Some studies reported that T stages, N stages, and clinical stages were factors influencing the prognosis of esophageal cancer patients[29,30]. This study conducted regular follow-up for patients and found that esophageal cancer patients receiving radiotherapy had an overall survival rate of 42.17%, and that the 3-year overall survival rate was markedly lower in patients with high serum Rpph1 expression than in those with low serum Rpph1 expression. Such findings suggest that Rpph1 is related to the prognosis of esophageal cancer patients.Rpph1 can therefore be used as a biomarker to diagnose esophageal cancer and evaluate prognosis.

    An extensive amount of literature has revealed that radiotherapy depends on ionizing radiation to induce tumor cell apoptosis, the occurrence of which is regulated by multiple genes[9,31,32]. Therefore, changes in apoptosis-related genes can accurately reflect cell sensitivity to radiotherapy. Previous studies have revealed that the esophageal cancer cell lines TE-1 and Kyse150 can effectively reflect radio-sensitivity.We found no contaminations in TE-1 and Kyse150 cell lines, so the detection of these two cell lines can provide a certain theoretical basis for the experiment[33,34]. To validate the role of Rpph1 on the radio-sensitivity of esophageal cancer cells, this study employed a colony formation assay to assess cell viability. The results of the colony formation assay demonstrated that Rpph1 silencing did not affect the colony sizes of TE-1 and Kyse150 cells. The cell-surviving fractions decreased with increasing irradiation doses in all transfection groups, with a stronger decrease in the siRNARpph1 group, suggesting that silencing Rpph1 expression can significantly enhance cell sensitivity to radiotherapy. Colony formation assays conducted by Jianget al[35]showed that downregulation of the lncRNA TUG1 increased the radio-sensitivity of bladder cancer cells by suppressing the expression of HMGB1. Whether Rpph1 uses the same mechanism of action requires further studies. One former study believed that radiation-induced apoptosis is one form of radiation-induced cell death[36]. The apoptosis experiments in this study revealed that apoptosis of the siRNA-Rpph1 group was much stronger than that of the blank control group and the siRNA-NC group when TE-1 and Kyse150 cells were exposed to irradiation at a dose of 4 Gy and 6 Gy, indicating that Rpph1 silencing can increase radiation-induced cell apoptosis.After radiotherapy, the siRNA-Rpph1+IR group exhibited ramped-up Bax and caspase-3 expression, and a reduction in Bcl-2 expression (anti-apoptotic protein),suggesting that Rpph1 silencing can intensify the radio-sensitivity of esophageal cancer cells by promoting cell apoptosis.

    Figure 2 Relationship between long non-coding RNA Rpph1 expression and clinicopathological features of esophageal cancer. A: Relationship between long non-coding RNA (lncRNA) Rpph1 and short-term response, T stage, N stage, and clinical stages; B: Diagnostic value of lncRNA Rpph1 for short-term response,T stage, N stage, and clinical stages. Note: b indicates bP < 0.001 when the two groups were compared.

    Cell cycle regulation may be critical for the radio-sensitivity of tumor cells[37].Radiation can induce DNA double-strand breaks and then lead to G2/M phase arrest and DNA damage repair, while G2/M phase arrest is a process of tumor cell selfrepair[38,39]. Zhanget al[20]claimed that Rpph1 may influence the progression of breast cancer by regulating cell proliferation and cell cycle. In this study, radiotherapy led to more G2/M phase cells. The siRNA-Rpph1+IR group (6 Gy) had significantly more G2/M phase cells than the siRNA-NC + IR group (P< 0.05), indicating that downregulation of Rpph1 expression can effectively alleviate radiation-induced G2/M phase arrest, thereby enhancing the radio-sensitivity of TE-1 and Kyse150 cells.To figure out the possible biological potential of Rpph1 in esophageal cancer, we silenced Rpph1 expression and discovered that Rpph1 silencing significantly inhibited the cell proliferation and migration of esophageal cancer. A recent study revealed that Rpph1 had a regulatory effect on the inflammation and proliferation of mesangial cells by stimulating the Gal-3/Mek/Erk pathway in diabetic nephropathy[40].However, the way Rpph1 regulates the biological behavior of esophageal cancer cells requires further studies. EMT is a basic biological process by which epithelial cells lose their polarity and transform into a mesenchymal phenotype[41]. EMT is associated with radio-sensitivity in esophageal cancer[41]. In this study, the expression levels of EMT-related proteins Vimentin and N-cadherin were greatly reduced, E-cadherin expression increased in the siRNA-Rpph1 group, and the decrease or increase of EMT-related proteins was sharper after radiotherapy. While we speculate that Rpph1 may be involved in the regulation of EMT, the specific mechanism needs to be confirmed by further research.

    Figure 3 Effect of long non-coding RNA Rpph1 on cancer cell sensitivity to radiotherapy. A: Expression of Rpph1 in esophageal cancer cell lines TE-1 and Kyse150 in different transfection groups; B: Role of Rpph1 on cell colonies; C: Effect of Rpph1 on the survival of TE-1 and Kyse150 cells after radiotherapy. bP <0.001 when the two groups were compared.

    This study is subject to certain limitations. The specific target genes or signaling pathways on which Rpph1 acts to regulate the biological function of esophageal cancer cells remains unknown. The function of tumor suppression by Rpph1 in esophageal cancer animal models needs to be further explored. In addition, the possibility of Rpph1 functioning as a target for treating esophageal cancer should be confirmed by abundant future studies.

    In summary, Rpph1 is highly expressed in esophageal cancer. To a certain extent,silencing Rpph1 expression can promote cell apoptosis, inhibit cell proliferation and migration, relieve radiation-induced G2/M phase arrest, and enhance radiosensitivity.

    Figure 4 Effect of long non-coding RNA Rpph1 on radiation-induced apoptosis and apoptosis proteins. A: Apoptosis of TE-1 cells after irradiation; B:Apoptosis of Kyse150 cells after irradiation; C: Comparison of apoptosis rate in esophageal cancer cells between different transfection groups; D: Expression of apoptosis-related proteins in esophageal cancer cells after irradiation. bP < 0.001 when the two groups were compared.

    Figure 5 Effect of long non-coding RNA Rpph1 on radiation-induced cell cycle. A: Changes in cell cycle of TE-1 cells; B: Changes in cell cycle of Kyse150 cells;C: Cell cycle distribution of TE-1 cells; D: Cell cycle distribution of Kyse150 cells.

    Figure 6 Effect of long non-coding RNA Rpph1 on radiation-induced cell proliferation, migration, and epithelial-mesenchymal transition. A: Cell growth curves of TE-1 and Kyse150 at different time points; B: Cell growth curves of TE-1 and Kyse150 at different radiotherapy doses; C: Comparison of cell migration of esophageal cancer cells; D: Expression of EMT-related proteins in esophageal cancer cells. Note: b indicates bP < 0.001 when the two groups were compared.

    ARTICLE HIGHLIGHTS

    Research background

    As a common digestive tract tumor, esophageal cancer is typically treated by radiotherapy. Poor responses to radiotherapy in most patients are prone to causing local radiotherapy failure. It is therefore essential to find new radiosensitizers to enhance the response of cancer cells to radiotherapy and increase the survival rate of esophageal cancer patients with radiation resistance. The long non-coding RNA (lncRNA) Rpph1 is highly expressed in human gastric cancer tissues, which also decreases breast cancer cell proliferation as well as tumorigenesis. In fact, the expression of lncRNA Rpph1 in esophageal cancer and its relationship with radiosensitivity have not been thoroughly investigated.

    Research motivation

    LncRNA Rpph1 is found abnormally expressed in a variety of cancers. The possibility that Rpph1 impacts the radio-sensitivity of esophageal cancer cells requires more research.

    Research objectives

    This study was intended to reveal the value of lncRNA Rpph1 in esophageal cancer as well as its effect on cell sensitivity to radiotherapy.

    Research methods

    We initially detected the expression of lncRNA Rpph1 in esophageal cancer tissue samples obtained surgically. Subsequently, siRNA-NC or siRNA-Rpph1 was transfected into esophageal cancer cell lines, and a blank control group was set where cells were not transfected with anything. Consequently, we analyzed the effect of Rpph1 on the biological behavior of esophageal cancer cells exposed to irradiation.

    Research results

    We found that Rpph1 is highly expressed in esophageal cancer. Rpph1 can be applied for the diagnosis of esophageal cancer and identify the pathological characteristics of patients. The results of 3-year survival supported patients with low Rpph1 expression over those with high Rpph1 expression (P < 0.05). Cytological studies indicated that silencing the expression of Rpph1 contributed to the enhancement of radio-sensitivity of esophageal cancer cells and cell apoptosis via regulating apoptosis-linked proteins, thus relieving the radiation-induced G2/M phase cell cycle arrest. It also helped restrain cell proliferation and migration and regulate the expression of epithelial-mesenchymal transition (EMT)-related proteins.

    Research conclusions

    Rpph1 is highly expressed in esophageal cancer. Silencing Rpph1 expression has an influence on the biological behavior of tumor cells and the enhancement of radio-sensitivity.

    Research perspectives

    This study revealed the value of Rpph1 in the diagnosis of esophageal cancer and concluded that silencing Rpph1 expression can enhance the radiotherapy of tumor cells, illuminating a new target for the future treatment of esophageal cancer.

    欧美激情高清一区二区三区| 又大又爽又粗| 精品国产亚洲在线| 久久久水蜜桃国产精品网| 久久精品亚洲av国产电影网| 大陆偷拍与自拍| 亚洲aⅴ乱码一区二区在线播放 | 国产在线一区二区三区精| 亚洲人成电影免费在线| 天堂中文最新版在线下载| 免费一级毛片在线播放高清视频 | 成人手机av| 国产精品久久久人人做人人爽| av不卡在线播放| 女警被强在线播放| 午夜免费鲁丝| 国产蜜桃级精品一区二区三区 | 国产单亲对白刺激| 国产亚洲精品久久久久久毛片 | 国产精品av久久久久免费| 国产主播在线观看一区二区| 夜夜夜夜夜久久久久| 一进一出好大好爽视频| 黄色成人免费大全| 免费av中文字幕在线| 欧美日韩成人在线一区二区| 日韩视频一区二区在线观看| 丰满的人妻完整版| 午夜福利一区二区在线看| 亚洲少妇的诱惑av| 国产欧美日韩一区二区三区在线| 亚洲成人免费av在线播放| 国产成+人综合+亚洲专区| 国产欧美亚洲国产| 一区二区三区激情视频| 女人被狂操c到高潮| 久久中文字幕一级| 日本欧美视频一区| 岛国在线观看网站| 欧美国产精品一级二级三级| 国产麻豆69| 欧美激情高清一区二区三区| 天堂动漫精品| 女人高潮潮喷娇喘18禁视频| 久久热在线av| 老司机午夜十八禁免费视频| 极品教师在线免费播放| 精品一品国产午夜福利视频| 国产精品影院久久| 女性被躁到高潮视频| 婷婷丁香在线五月| 久久久国产成人免费| 国产av精品麻豆| 午夜福利免费观看在线| 丰满的人妻完整版| 国产不卡一卡二| 黄色 视频免费看| 少妇猛男粗大的猛烈进出视频| 可以免费在线观看a视频的电影网站| videosex国产| 国产亚洲精品第一综合不卡| 精品少妇一区二区三区视频日本电影| 在线播放国产精品三级| 欧美人与性动交α欧美精品济南到| 男女之事视频高清在线观看| 久久精品aⅴ一区二区三区四区| 黄色片一级片一级黄色片| а√天堂www在线а√下载 | 国产精品综合久久久久久久免费 | 国产单亲对白刺激| 色精品久久人妻99蜜桃| 国产av一区二区精品久久| 久久久国产欧美日韩av| 丰满饥渴人妻一区二区三| 搡老熟女国产l中国老女人| 一区二区三区国产精品乱码| 精品久久久久久久久久免费视频 | 91精品三级在线观看| 19禁男女啪啪无遮挡网站| 亚洲精品粉嫩美女一区| 黑人巨大精品欧美一区二区mp4| 看免费av毛片| 丰满迷人的少妇在线观看| www.熟女人妻精品国产| 黑人猛操日本美女一级片| av片东京热男人的天堂| 亚洲熟女精品中文字幕| 久久九九热精品免费| 国产蜜桃级精品一区二区三区 | www.999成人在线观看| 欧美激情 高清一区二区三区| 巨乳人妻的诱惑在线观看| 免费看a级黄色片| 波多野结衣av一区二区av| 国产一区二区三区视频了| 曰老女人黄片| 一边摸一边抽搐一进一出视频| 亚洲自偷自拍图片 自拍| 91九色精品人成在线观看| 色综合婷婷激情| 91精品国产国语对白视频| 啦啦啦在线免费观看视频4| 久久ye,这里只有精品| 亚洲综合色网址| 色婷婷av一区二区三区视频| 不卡av一区二区三区| 亚洲情色 制服丝袜| av电影中文网址| 老司机亚洲免费影院| 美女国产高潮福利片在线看| 一级黄色大片毛片| 精品国内亚洲2022精品成人 | 丝瓜视频免费看黄片| 一级a爱视频在线免费观看| 中文字幕av电影在线播放| 精品国产乱子伦一区二区三区| 熟女少妇亚洲综合色aaa.| 首页视频小说图片口味搜索| 欧美日韩福利视频一区二区| 女警被强在线播放| 亚洲精品美女久久久久99蜜臀| 成人av一区二区三区在线看| 日韩中文字幕欧美一区二区| 午夜福利影视在线免费观看| 精品熟女少妇八av免费久了| 久久久久国内视频| 色94色欧美一区二区| 色在线成人网| 悠悠久久av| 动漫黄色视频在线观看| 久久精品国产综合久久久| 日日夜夜操网爽| 国产片内射在线| 久9热在线精品视频| 欧美日韩乱码在线| www.自偷自拍.com| 欧美国产精品va在线观看不卡| 欧美丝袜亚洲另类 | 热99re8久久精品国产| 国产精华一区二区三区| 欧美日韩一级在线毛片| 无人区码免费观看不卡| 99热网站在线观看| 天天添夜夜摸| 亚洲,欧美精品.| 国产视频一区二区在线看| 亚洲国产欧美网| 久久人妻av系列| 两个人看的免费小视频| 制服人妻中文乱码| 久久久久精品国产欧美久久久| 国产精品久久电影中文字幕 | 久久午夜综合久久蜜桃| 国产精品永久免费网站| 成年女人毛片免费观看观看9 | 9色porny在线观看| 国产亚洲精品一区二区www | 国产极品粉嫩免费观看在线| av有码第一页| 天天影视国产精品| 婷婷精品国产亚洲av在线 | 日韩人妻精品一区2区三区| 亚洲精华国产精华精| 一级,二级,三级黄色视频| 欧美在线一区亚洲| 1024视频免费在线观看| 一级作爱视频免费观看| 操美女的视频在线观看| 1024视频免费在线观看| 欧美日韩成人在线一区二区| 久久精品国产亚洲av高清一级| 免费观看人在逋| 十八禁网站免费在线| 交换朋友夫妻互换小说| 欧美日韩亚洲高清精品| av一本久久久久| 在线看a的网站| 午夜影院日韩av| 亚洲专区字幕在线| 免费不卡黄色视频| 精品人妻在线不人妻| 国产精品亚洲一级av第二区| 50天的宝宝边吃奶边哭怎么回事| 欧美黄色淫秽网站| av超薄肉色丝袜交足视频| 亚洲熟妇熟女久久| 99国产极品粉嫩在线观看| 岛国在线观看网站| 亚洲欧洲精品一区二区精品久久久| 香蕉国产在线看| 成人亚洲精品一区在线观看| 一级a爱片免费观看的视频| 亚洲专区国产一区二区| 午夜日韩欧美国产| 又大又爽又粗| 十八禁人妻一区二区| 高清av免费在线| 亚洲成a人片在线一区二区| av片东京热男人的天堂| 欧美一级毛片孕妇| 婷婷丁香在线五月| 老司机福利观看| 三级毛片av免费| 99久久精品国产亚洲精品| 国产精品亚洲av一区麻豆| 亚洲免费av在线视频| 欧美另类亚洲清纯唯美| 法律面前人人平等表现在哪些方面| 国产精品免费大片| 国产亚洲欧美精品永久| 欧美中文综合在线视频| 多毛熟女@视频| 久久午夜综合久久蜜桃| 亚洲 欧美一区二区三区| 在线国产一区二区在线| 一本综合久久免费| netflix在线观看网站| 一本综合久久免费| 午夜激情av网站| 老司机深夜福利视频在线观看| 亚洲欧美精品综合一区二区三区| 国产视频一区二区在线看| 久久人人97超碰香蕉20202| 天堂俺去俺来也www色官网| 日韩免费av在线播放| 国产不卡一卡二| 一进一出好大好爽视频| 啦啦啦 在线观看视频| 天堂中文最新版在线下载| 欧美色视频一区免费| 亚洲一区高清亚洲精品| 午夜成年电影在线免费观看| 丝袜美足系列| av免费在线观看网站| 国产一区二区三区视频了| 国产欧美日韩一区二区三区在线| 午夜福利乱码中文字幕| 国产一区在线观看成人免费| 亚洲第一av免费看| 1024香蕉在线观看| 黄色视频,在线免费观看| 男人的好看免费观看在线视频 | 夫妻午夜视频| 国产日韩一区二区三区精品不卡| 热re99久久国产66热| 黄色怎么调成土黄色| 黄色a级毛片大全视频| 黄色怎么调成土黄色| 亚洲欧美一区二区三区久久| 熟女少妇亚洲综合色aaa.| 两人在一起打扑克的视频| 人人澡人人妻人| 国内久久婷婷六月综合欲色啪| 精品欧美一区二区三区在线| 午夜久久久在线观看| 国产成+人综合+亚洲专区| 99精品久久久久人妻精品| 丝袜美腿诱惑在线| 叶爱在线成人免费视频播放| 啦啦啦 在线观看视频| 日韩 欧美 亚洲 中文字幕| www.自偷自拍.com| 成人18禁在线播放| 亚洲av第一区精品v没综合| 老汉色∧v一级毛片| 超碰成人久久| 久久人妻av系列| 久久热在线av| 在线av久久热| www.精华液| 黑人欧美特级aaaaaa片| 国产精品永久免费网站| 欧美精品av麻豆av| 亚洲欧洲精品一区二区精品久久久| 黄片播放在线免费| 侵犯人妻中文字幕一二三四区| 搡老岳熟女国产| 人成视频在线观看免费观看| 成人18禁在线播放| 在线观看www视频免费| 午夜福利欧美成人| 久久人妻福利社区极品人妻图片| 免费在线观看亚洲国产| av电影中文网址| 啦啦啦免费观看视频1| 男女免费视频国产| 午夜成年电影在线免费观看| 国产亚洲精品久久久久久毛片 | 日本a在线网址| 欧美一级毛片孕妇| 91在线观看av| 午夜福利乱码中文字幕| 中国美女看黄片| 最近最新中文字幕大全电影3 | 国产成+人综合+亚洲专区| 国产成人系列免费观看| 亚洲av欧美aⅴ国产| 久久午夜综合久久蜜桃| 午夜视频精品福利| 黑丝袜美女国产一区| 欧美一级毛片孕妇| 欧美精品亚洲一区二区| 悠悠久久av| 久久久久久久午夜电影 | 女人被狂操c到高潮| 无限看片的www在线观看| 啦啦啦免费观看视频1| 久久人妻福利社区极品人妻图片| 成熟少妇高潮喷水视频| 午夜激情av网站| 免费在线观看完整版高清| 欧美精品人与动牲交sv欧美| 欧洲精品卡2卡3卡4卡5卡区| 在线观看免费高清a一片| 国产精品久久久av美女十八| 99精品欧美一区二区三区四区| 中文字幕人妻丝袜制服| 亚洲成国产人片在线观看| 两性午夜刺激爽爽歪歪视频在线观看 | 国产欧美日韩精品亚洲av| 女警被强在线播放| 一本综合久久免费| 国产主播在线观看一区二区| 99热只有精品国产| 亚洲黑人精品在线| √禁漫天堂资源中文www| 麻豆成人av在线观看| 午夜福利一区二区在线看| 久久性视频一级片| 精品高清国产在线一区| 十八禁高潮呻吟视频| 国产精品av久久久久免费| e午夜精品久久久久久久| 欧美人与性动交α欧美软件| 午夜免费成人在线视频| 黑丝袜美女国产一区| 亚洲精品乱久久久久久| 97人妻天天添夜夜摸| 亚洲精品久久成人aⅴ小说| 久久精品国产亚洲av高清一级| 免费观看人在逋| 亚洲av欧美aⅴ国产| aaaaa片日本免费| 在线国产一区二区在线| 免费在线观看影片大全网站| 一进一出好大好爽视频| 色精品久久人妻99蜜桃| 国产日韩一区二区三区精品不卡| 一级a爱视频在线免费观看| 国产真人三级小视频在线观看| 一夜夜www| 两人在一起打扑克的视频| 色综合婷婷激情| 三上悠亚av全集在线观看| 国产不卡av网站在线观看| 日韩人妻精品一区2区三区| 亚洲精品国产一区二区精华液| 亚洲一区二区三区不卡视频| av天堂在线播放| 高清在线国产一区| 99热网站在线观看| 欧美日韩国产mv在线观看视频| www.自偷自拍.com| 91成年电影在线观看| 桃红色精品国产亚洲av| 亚洲情色 制服丝袜| 一二三四在线观看免费中文在| 久久狼人影院| 免费人成视频x8x8入口观看| 中出人妻视频一区二区| 99国产精品一区二区三区| 天天影视国产精品| 亚洲国产精品一区二区三区在线| 精品欧美一区二区三区在线| 午夜91福利影院| 大型黄色视频在线免费观看| 少妇被粗大的猛进出69影院| 午夜亚洲福利在线播放| 久久精品国产99精品国产亚洲性色 | 国产精品久久电影中文字幕 | 天天躁日日躁夜夜躁夜夜| 男人舔女人的私密视频| 母亲3免费完整高清在线观看| 精品高清国产在线一区| 国产欧美日韩精品亚洲av| 亚洲综合色网址| 身体一侧抽搐| 亚洲av熟女| 黄片播放在线免费| av中文乱码字幕在线| 一级片免费观看大全| 国产精品一区二区免费欧美| 亚洲 国产 在线| 精品国产乱码久久久久久男人| 国产片内射在线| 亚洲五月色婷婷综合| 久久久久精品人妻al黑| 免费在线观看亚洲国产| 国产精品久久久久成人av| 亚洲精品久久成人aⅴ小说| 中文字幕人妻熟女乱码| 久久精品亚洲熟妇少妇任你| 亚洲,欧美精品.| 很黄的视频免费| 又黄又粗又硬又大视频| 久久精品国产亚洲av香蕉五月 | 我的亚洲天堂| 久久人人97超碰香蕉20202| 香蕉丝袜av| 午夜久久久在线观看| 又紧又爽又黄一区二区| 亚洲久久久国产精品| 午夜精品久久久久久毛片777| 丝袜美足系列| 国产精品久久久av美女十八| 天堂中文最新版在线下载| av天堂久久9| av天堂在线播放| 亚洲七黄色美女视频| 欧美大码av| 999久久久精品免费观看国产| 精品国产乱码久久久久久男人| 黄色丝袜av网址大全| 国产深夜福利视频在线观看| 51午夜福利影视在线观看| 国产精品 欧美亚洲| 国产亚洲一区二区精品| 在线天堂中文资源库| e午夜精品久久久久久久| 亚洲精品一卡2卡三卡4卡5卡| av有码第一页| 91麻豆av在线| 丰满饥渴人妻一区二区三| 国产午夜精品久久久久久| 国产精品亚洲av一区麻豆| 9热在线视频观看99| 热99久久久久精品小说推荐| 在线观看免费午夜福利视频| 91九色精品人成在线观看| 1024香蕉在线观看| 亚洲人成伊人成综合网2020| 亚洲国产精品合色在线| 久久精品国产清高在天天线| 99久久国产精品久久久| a级毛片黄视频| 村上凉子中文字幕在线| 中亚洲国语对白在线视频| 精品卡一卡二卡四卡免费| 国产av一区二区精品久久| 很黄的视频免费| 欧美成人午夜精品| 一个人免费在线观看的高清视频| 黑人猛操日本美女一级片| 99久久人妻综合| 大香蕉久久成人网| 亚洲av片天天在线观看| 高清视频免费观看一区二区| 国产高清激情床上av| 午夜福利在线观看吧| 色综合婷婷激情| 午夜免费观看网址| 在线av久久热| 悠悠久久av| 亚洲精品自拍成人| 色播在线永久视频| 久久亚洲精品不卡| bbb黄色大片| 国产精品免费视频内射| 大陆偷拍与自拍| 亚洲精品久久午夜乱码| 久久国产精品男人的天堂亚洲| 久久性视频一级片| 无限看片的www在线观看| 人人妻人人爽人人添夜夜欢视频| 操美女的视频在线观看| 天堂俺去俺来也www色官网| 国产有黄有色有爽视频| 人妻丰满熟妇av一区二区三区 | 老汉色av国产亚洲站长工具| 婷婷丁香在线五月| 老熟女久久久| 制服诱惑二区| 亚洲va日本ⅴa欧美va伊人久久| 人妻一区二区av| 老司机午夜福利在线观看视频| 国产欧美日韩一区二区三区在线| 国产精品久久视频播放| 国产成+人综合+亚洲专区| www.999成人在线观看| 亚洲avbb在线观看| ponron亚洲| 亚洲人成电影观看| 一本综合久久免费| 天堂√8在线中文| 老司机福利观看| 成人av一区二区三区在线看| 高清黄色对白视频在线免费看| 人人妻人人添人人爽欧美一区卜| 国产一区有黄有色的免费视频| 在线观看免费午夜福利视频| 777久久人妻少妇嫩草av网站| av有码第一页| 两个人看的免费小视频| avwww免费| 免费不卡黄色视频| 飞空精品影院首页| a在线观看视频网站| 99国产精品一区二区三区| 久久久久久久午夜电影 | av网站在线播放免费| 久久婷婷成人综合色麻豆| 久久精品91无色码中文字幕| 少妇猛男粗大的猛烈进出视频| 日本精品一区二区三区蜜桃| 国产aⅴ精品一区二区三区波| 美女午夜性视频免费| 操美女的视频在线观看| 动漫黄色视频在线观看| 在线看a的网站| 看黄色毛片网站| 美女高潮喷水抽搐中文字幕| 成熟少妇高潮喷水视频| 亚洲伊人色综图| 香蕉国产在线看| 欧美精品亚洲一区二区| 天天添夜夜摸| 精品国产乱子伦一区二区三区| 久热这里只有精品99| 中出人妻视频一区二区| 亚洲精品在线观看二区| 免费在线观看黄色视频的| 欧美日韩亚洲高清精品| 久久久国产成人免费| 精品电影一区二区在线| 香蕉国产在线看| 一边摸一边做爽爽视频免费| 51午夜福利影视在线观看| 一本综合久久免费| 精品国产一区二区三区久久久樱花| 一区二区三区国产精品乱码| 国产午夜精品久久久久久| 久久影院123| 久久中文看片网| 久久国产精品人妻蜜桃| 美女午夜性视频免费| 91麻豆av在线| 国产又色又爽无遮挡免费看| 国产97色在线日韩免费| 老熟女久久久| aaaaa片日本免费| 国产野战对白在线观看| 夜夜夜夜夜久久久久| 日本五十路高清| 国产国语露脸激情在线看| 丝袜在线中文字幕| 欧美日韩成人在线一区二区| 日本黄色视频三级网站网址 | 黑人操中国人逼视频| 露出奶头的视频| 国产激情欧美一区二区| 人人妻人人爽人人添夜夜欢视频| 国产日韩一区二区三区精品不卡| 国产高清激情床上av| 老司机福利观看| 日韩成人在线观看一区二区三区| 国产亚洲欧美98| 精品亚洲成a人片在线观看| 国产精品秋霞免费鲁丝片| 亚洲欧美激情在线| 欧美精品人与动牲交sv欧美| 欧美 亚洲 国产 日韩一| 欧美日韩亚洲综合一区二区三区_| 精品高清国产在线一区| 精品人妻熟女毛片av久久网站| 国产aⅴ精品一区二区三区波| 搡老岳熟女国产| 久久草成人影院| 日本欧美视频一区| 777米奇影视久久| 亚洲片人在线观看| 91麻豆av在线| 国产又爽黄色视频| 校园春色视频在线观看| 女人高潮潮喷娇喘18禁视频| 香蕉国产在线看| 国产亚洲精品久久久久久毛片 | 两性午夜刺激爽爽歪歪视频在线观看 | 黄色片一级片一级黄色片| 亚洲精品中文字幕在线视频| 99riav亚洲国产免费| 国产精品亚洲一级av第二区| av网站免费在线观看视频| 成人永久免费在线观看视频| 国产av精品麻豆| 丝袜美足系列| 极品教师在线免费播放| 又黄又爽又免费观看的视频| 亚洲av欧美aⅴ国产| 免费日韩欧美在线观看| 欧美日韩亚洲综合一区二区三区_| 亚洲专区字幕在线| 91av网站免费观看| 国产在视频线精品| 久久99一区二区三区| 美女国产高潮福利片在线看| 高清毛片免费观看视频网站 | 日韩欧美在线二视频 | 夫妻午夜视频| 午夜福利一区二区在线看| 黑人欧美特级aaaaaa片| 中文字幕人妻丝袜一区二区| 制服人妻中文乱码| 91麻豆精品激情在线观看国产 | 多毛熟女@视频| 国产主播在线观看一区二区|