| nasopharyngeal carcinoma is one of the most common nonlymphocytic malignant tumor, which is associated with Epstein-Barr Virus (EBV). nasopharyngeal carcinoma is common in southeast Asia particularly in southern China,where is reported an annual incidence of 100/100,000 as compared to the wordwide incidence of 1/100,000. Currently, the clinical treatment of nasopharyngeal carcinoma although the means are more, including radiotherapy, molecular targeted therapy, effects are good also. Most nasoph-ryngeal carcinoma patients were diagnosed at the late stages, owing to the deep location of nasopharynx, small primary tumor and vague symptoms of the disease. In contrast to other head and neck cancer, nasopharyngeal carcinoma is prone to invade and metas-tasize because of its characteristic and abundant peripheral lymphoid tissues. nasopharyngeal carcinoma patients has distant metastasis earlier than the presence of clinical symptoms.Approximately 90% of patients with the initial diagnosed nasopharyngeal carcinoma present with Cervical lymph nodes. Thus, an improved understanding of the molecular mechanisms involved in the invasion and metastasis of nasopharyngeal carcinoma will lead to improved treatments and prognosis factors for nasopharyngeal carcinoma patients.Normal tissue cells in special cases, with migration, such as embryonic development, wound healing, immune cells. The tumor cells from normal cells through the evolution of the formation of stronger than normal tissue cell migration and proliferation. Although tumor cells have their own characteristics, as derived from normal tissue cells, in many ways still in use, or enhance existing features. We are trying to find has been confirmed in normal cells is closely related with cell proliferation and migration factors, and to study its mechanism.TRP channels is a non-selective cation channel superfamily, first identified in Drosophila species. On the bases of homology and channel function, the TRP family is divided into seven main subfamilies:TRPA(anktmi),TRPC (canonical TRP) TRPM (melastatin, long-TRP),TRPML (mucolipin),TRPN (nompc),TRPP (polycystin),TRPV (vanilloid). TRP channels are widely distributed family members, now known, in the central nervous system, peripheral nervous system, skin, cardiovascular system, respiratory system, gastrointestinal system, urinary and reproductive systems, and immune and endocrine systems. TRPC C-terminal to TRP box as a starting point, there is a conserved amino acid sequences of the 25 areas, called the TRP domain, the TRP domain is currently unclear, but other studies suggest Rohacs, TRP TRP channel domains involved in the regulation features.TRPC1 in the brain, heart, kidney, lung, muscle, prostate, skin, testis and ovary were detected in its high level of expression. As the first mammalian TRP channels are found, TRPC1 recognized function is involved in receptor-mediated, calcium-dependent smooth muscle contraction and glandular secretion and function of membrane receptors involved in activation of phospholipase C (PLC)-mediated after of calcium ions to enter.TRPC1, a member of the TRPC subfamily, is a non-selective cation channel with predominant permeability for Ca2+ and Mg2+TRP channels regulate intracellular Ca2+ concentrations by acting as Ca2+ entry pathways in the plasma membrane, Ca2+ signals in the body associated with many important physiological processes such as cell growth and cell necrosis, dendritic formation, cell cycle regulation and cell migration. A large number of studies have shown that intracellular calcium is closely related with the tumor, directly involved in the regulation of tumor growth, invasion, metastasis and differentiation, leading to uncontrolled tumor proliferation and abusethen result in enhanced proliferation, aberrant differentiation, and impaired ability to die. Yassine El Hiani et have reported that high [Ca2+]o leads to TRPC1 over-expression and cell proliferation. Valerie C.et has shown TRPC1 regulates migration in malignant gliomas.Recent studies have suggested more and more importance for TRPC1 in the processes of cell migration and proficeation.Our study aimed to explore the possibility of TRPC1 as the molecular target of nasopharyngeal carcinoma and lay the potent theoretical basis on diagnosis, prognosis evaluation and combined therapy design of nasopharyngeal carcinoma patients.Methods1. The distributions and expressions of TRPC1 protein in human normal nasopharyngeal tissues and nasopharyngeal carcinomaSelected 30 cases of nasopharyngeal carcinoma biopsy, normal nasopharyngeal biopsy in 20 cases。inmunohistochemistry were used to examine The distributions and expressions of TRPC1 protein in human normal nasopharyngeal tissues and nasopharyngeal carcinoma2. The expressions of TRPC1 gene in human nasopharyngeal carcinoma cellsReal time PCR and immunohistochemistry were used to examine the expressions of TRPC1 gene in human 3 nasopharyngeal carcinoma cell lines named CNE-2,C666-1 and 6-10B。According to the gene TRPC1 and the housekeeping geneβ-actin amplification curve, get their Ct value calculated by the formula 2-ΔΔCt value, in the relative quantitative analysis, we use 2-ΔΔCt said that compared with the CNE2,6-10B and C666-1 cell lines relative gene expression levels of TRPC1. Single sample t test to compare 6-10B,C666-1 cell line and CNE2 in TRPC1 mRNA levels, independent sample t test to compare the 6-10B and C666-1 cell lines TRPC1 mRNA levels3. All siRNA duplexes were synthesized by Shanghai GenePharma Co. In the wells, three TRPC1 siRNA or negative control siRNA, were then transiently transfected into CNE-2 nasopharyngeal carcinoma cell line. Real time PCR were performed to detect the interference efficiency of TRPC1 gene. The highest interference efficiency was used to construct CNE2-siTRPCl stably cell lines.4. CNE-2 cells were transfected with the pSuper-retro-puro construct and empty construct by using Lipofectamine 2000 and selected for stable transfectants by zeocin treatment. Real time PCR and Western blot were performed to detect the expression of TRPC1 gene/protein.5,TRPC1 gene silencing on changes of TRPC1,MMP2 and MMP9 were detected by QPCR in vitro.According to the gene TRPC1 and the housekeeping geneβ-actin amplification curve, get their Ct value calculated by the formula 2-ΔΔCt values。In the relative quantitative analysis to 2-ΔΔCt Ct represents the untransfected CNE2 compared to interference (CNE2-siTRPC1) and load (CNE2-vetor) transfected cell lines CNE2 TRPC1, MMP2 and The relative gene expression levels of MMP9。Single sample t test to compare CNE2-siTRPC1, CNE2-vetor and CNE2 cells, TRPC1, MMP2 and MMP9 mRNA levels, independent sample t test to compare CNE2-siTRPC1 and CNE2-vetor cells TRPC1, MMP2 and MMP9mRNA level.6,The effect of TRPC1 gene silencing on the biological characters of nasopharyngeal carcinoma cells.(1) The effects of TRPCl gene silencing on cell invasion were detected by invasive chamber in vitro.(2) The effects of TRPC1 gene silencing on cell proliferation in vitro were detected by MTT method and flow cytometry.7,TRPC1 inhibitors on the biological characteristics of nasopharyngeal carcinoma cellsAfter Using the TRP channel inhibitors 2-APB acts on CNE2 cells,Real time PCR and Western blot were performed to detect the expression of TRPC1 gene/protein. And changes of MMP2 and MMP9 were detected by QPCR in vitro.The effects of TRPC1 gene on cell proliferation in vitro were detected by MTT method and flow cytometry, while those on cell migration and invasion were detected by invasive chamber in vitro.Results1. The distributions and expressions of TRPC1 protein in human normal nasopharyngeal tissues and nasopharyngeal carcinoma tissues.Immunohistochemistry showed high expression in nasopharyngeal carcinoma and normal tissue with low expression. TRPCl protein in 30 cases of nasopharyngeal carcinoma, the positive expression rate of the total 90% (27/30), high expression rate was 80%(24/30). TRPC1 protein expression in nasopharyngeal carcinoma (90%) was significantly higher than that of the normal nasopharyngeal tissues (5.0%) (χ2=22.005, P=0.000<0.05). TRPC1 protein expression in nasopharyngeal carcinoma was significantly higher than normal tissues (Z=-5.274, P=0.000<0.05).2. The expressions of TRPC1 gene in human nasopharyngeal carcinoma cellsTRPC1 gene expressions in 3 human NP carcinoma cell lines were detected by real time PCR. The results showed that Results of fluorescent quantitative PCR Comparing with CNE2-siTRPC1 (2-ΔΔCt=1),the expression of mRNA in 6-10B (2-ΔΔCt=0.552±0.037)和C666-1 (2-ΔΔCt=0.736±0.095) was downregulated (t =-20.918,-4.757, P=0.002,0.041). Positive signals of TRPC1 expression were observed in cytoplasm human nasopharyngeal carcinoma cell line by immunocytochecmistry.3. QPCR analyses showed that, within 48 h after transfection of CNE2 cells with TRPC1 siRNA No.1-3, the expression of TRPC1 protein was suppressed 80%. Finally, the motility of TRPC1 knockdown cells was analyzed using the transwell chamber assay and MTT (named CNE2-siTRPC 1 cell)4.Retroviruses were produced using the vector(pSuper-retro-siTRPC1,pSuper-retro PIK) and 293FT packaging cell line by calcium phosphate.Collect the cultur e after 48h.Retroviruses were used to infect CNE2 cell,then puromycin-resistant cells were pooled.A stable cell line with a persistent silencing of TRPC1 and control was established, which was named as CNE2 -siTRPC1,.CNE2-vetor,5. Results of fluorescent quantitative PCR Comparing with CNE2 (2-ΔΔCt=1) and CNE2-vetor (2-ΔΔCt=0.884±0.124),the expression of TRPC1mRNA in CNE2-siTRPC1 (2-ΔΔCt=0.149±0.031) was downregulated (t=48.125,P=0.000; t=-9.979, P<0.001). Results of western blot The expression of TRPC1 protein in CNE2-siTRPC1 was downregulated comparing CNE2-vetor and CNE2 (F=17057.026, P<0.001)6. TRPC1 gene silencing on changes of MMP2 and MMP9 were detected by QPCR in vitro.(1)MMP2 gene expressions in human NP carcinoma cell lines (CNE2-siTRPC1,CNE2 and CNE2-vetor) were detected by real time PCR. The results showed that Results of fluorescent quantitative PCR Comparing with CNE2 (2-ΔΔCt=1) and CNE2-vetor (2-ΔΔCt= 0.953±0.054),the expression of MMP2 mRNA in CNE2-siTRPC1 (2-ΔΔCt= 0.251±0.019) was downregulated(t=-66.090,P=0.000; t=-21.174, P<0.001)(2)MMP9 gene expressions in 3 human NP carcinoma cell lines(CNE2-siTRPC1,CNE2 and CNE2-vetor) were detected by real time PCR. The results showed that Results of fluorescent quantitative PCR Comparing with CNE2 (2-ΔΔCt=1) and CNE2-vetor (2-ΔΔCt= 0.942±0.011),the expression of MMP9mRNA in CNE2-siTRPC1(2-ΔΔCt= 0.264±0.011) was downregulated (t=-115.678,P=0.000; t=-20.845, P=0.002)7. TRPC1 gene silencing on the biological characteristics of nasopharyngeal carcinoma cells(1)The changes of cell invasive abilities after TRPCl gene silencing were detected by invasive chamber experiment in vitro and the results showed that compared to CNE2 and CNE2-vetor cell, the invasive abilities of CNE-2-siTRPC1 cell decreased obviously (F=9.289, P=0.015), suggesting that TRPC1 silencing inhibited the invasive abilities of nasopharyngeal carcinoma cell. (2)MTT method was carried out to observe the cell proliferation in vitro after TRPC1 gene silencing. Compared to CNE-2 cell line, CNE-2-siTRPC1cell line had slower proliferative rate in the time-dependent manner (F= 574.752, P=0.000). The cell cycle of CNE-2-siTRPC1 was obviously reduced compare with CNE-2 by flow cytometry (F= 9.648, P=0.013), indicating that cell proliferation became slowly after TRPC1 gene silencing. The results showed that growth in vitro of tumor cells were obviously inhibited after the expressive level of TRPC1 decreased.8. TRPC1 inhibitors on the biological characteristics of nasopharyngeal carcinoma cells(1) The changes of TRPC1 gene/proteins after 2-APB were detected by QPCR and western blotResults of fluorescent quantitative PCR:CNE2-2-APB cells, CNE2-siTRPC1 cells, CNE2 and CNE2-vetor cells TRPC1mRNA There were significant differences in the overall (F=31.799, P=0.001) Comparing with CNE2 (2-ΔΔCt=1) and CNE2-vetor (2-ΔΔCt=0.884±0.124),the expression of TRPC1mRNA in CNE2-siTRPC1 (2-ΔΔCt=0.149±0.031) and CNE2-2-APB (2-ΔΔCt=0.884±0.124) was downregulated(t=-17.065,P=0.003; t=-10.068,P=0.010). Results of western blot: Respectively, from CNE2, CNE2-vetor, CNE2-2-APB and CNE2-siTRPC1 total protein extract detected by Western blot showed that CNE2-2-APB cells, CNE2-siTRPC1 cells, CNE2 cells and CNE2-vetor The TRPC1mRNA There were significant differences in overall. the expression of TRPC1 protein in CNE2-siTRPC1 and CNE2-2-APB was downregulated (F=92.022,P=0.000).The expression of TRPC1 protein in CNE2-siTRPC1 was downregulated comparing CNE2-vetor and CNE2 (P=0.002; P=0.002)(2) TRPC1 was detected by QPCR after blocking the MMP2 and MMP9 in vitro situation.Quantitative PCR results:CNE2-2-APB cells, CNE2-siTRPC1 cells, CNE2 and CNE2-vetor (2-ΔΔCt=1.043±0.073) cells MMP2mRNA overall there was a significant difference (F=148.035, P=0.001), CNE2-2-APB (0.310±0.389) and CNE2-siTRPC1 cells (2-ΔΔCt=0.235±0.010) and as control of cell CNE2 (2-ΔΔCt-1) compared to, MMP2 mRNA levels was significantly lower (t=-30.478, P= 0.000; t =-129.978, P=0.000). Similarly, MMP9 fluorescence quantitative PCR results showed that:CNE2-2-APB cells, CNE2-siTRPC1 cells, CNE2 and CNE2-vetor cells MMP9mRNA overall there was a significant difference (F=386.614, P=0.000), CNE2-2-APB and CNE2-siTRPC1 cells (2-ΔΔCt-0.149±0.031) and as control of cell CNE2 (2-ΔΔCt=1) compared to, MMP9 mRNA levels were significantly lower (t =-55.693, P=0.000; t=-93.595, P=0.000).Similarly, MMP9 fluorescence quantitative PCR results showed that:CNE2-2-APB cells, CNE2-siTRPC1 cells, CNE2 and CNE2-vetor cells MMP9mRNA overall there was a significant difference (F=386.614, P=0.000), CNE2-2-APB and CNE2-siTRPC1 cells (2-ΔΔCt= 0.149±0.031) and as control of cell CNE2 (2-ΔΔCt=1) compared to, MMP9 mRNA levels were significantly lower (t=-55.693, P=0.000; t=-93.595, P=0.000).(3) MTT method was carried out to observe the cell proliferation in vitro after 2-APB. Compared to CNE-2 celland CNE2-vetor cell line,2-APB-CNE-2 cell line had slower proliferative rate in the time-dependent manner (F=393.584,P=0.000).The cell cycle of 2-APB-CNE-2 cell was obviously reduced compare with CNE-2 by flow cytometry, indicating that cell proliferation became slowly after 2-APB. Significant difference (F=14.010, P=0.002<0.05), G1 phase was increased significantly (F=25.960, P=0.000), showed that after the TRPC1 channel blockers on cell cycle arrest in Gl.The results showed that growth in vitro of tumor cells were obviously inhibited after 2-APB。(4)After TRPC1 channels were inhibited by specific ion channel inhibitor 2-APB.The changes of cell migration abilities after were detected by invasive chamber experiment in vitro and the results showed that migration abilities of nasopharyngeal carcinoma cell were difference obviously (F=686.820,P=0.000) among four cell lines, compared to CNE-2 cell and CNE2-siTRPCl cell, the migration abilities of CNE-2-2-APB cell decreased obviously (P=0.000,P=0.000), suggesting that 2-APB inhibited the migration abilities of nasopharyngeal carcinoma cell.(5)After TRPC1 channels were inhibited by specific ion channel inhibitor 2-APB.The changes of cell invasive abilities after were detected by invasive chamber experiment in vitro and the results showed that invasive abilities of nasopharyngeal carcinoma cell were difference obviously (F=434.989,P=0.000) among four cell lines, compared to CNE-2 cell and CNE2-siTRPC1 cell, the invasive abilities of CNE-2-2-APB cell decreased obviously (P=0.000,P=0.001), suggesting that 2-APB inhibited the invasive abilities of nasopharyngeal carcinoma cell.Conclusions1. TRPC1 protein was highly expressed in tumors and it can be acted as one important tumor maker for tumorigenesis and development;2. TRPC1 stably expressing cell line CNE2-siTRPC1 was established with pSuper-retro-puro-siTRPCl transfection and puro selection. which would be provided a valuable tool for TRPC1 functional studies;3. TRPC1 gene silencing and TRP inhibitors can inhibit the proliferation and invasion ofnasopharyngeal carcinoma cell, indicating TRPC1 can promote the proliferation and invasion of nasopharyngeal carcinoma cell and suggesting the important role of TRPC1 gene in proliferation and invasion of nasopharyngeal carcinoma cell.Innovations of our study:1. TRPC1 gene had relationship with proliferation, invasion and prognosis of nasopharyngeal carcinoma, which laid theoretical basis on targeted therapy of nasopharyngeal carcinoma;2. The establishment of a nasopharyngeal carcinoma cell line with TRPC1 stable silencing will be provide a valuable tool for TRPC1 functional studies.
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