Effects Of Silencing Id-1 On Human Oral Cancer Cells

SectionⅠExpression of Id-1 gene in oral cancer cellsObjectiveOral cancer is more common clinical disease, the incidence of an increasing trend in recent years, their age of onset of 40 to 60 years as the peak onset age, but 60 to 70-year-old age group a marked increase in the incidence, accounting for 17.8%, the average age of onset 54.1 years old, showed that the incidence of oral cancer has an aging trend. Most oral cancers occur in the oral cavity (excluding lip), accounting for 78.4% of the total, followed by the major salivary glands, maxillary sinus, neck, throat, lips. Mouth predilection sites in sequence as the tongue, upper and lower gums, cheek, palate, upper and lower jaw, floor of the mouth, tongue cancer has leapt to the top. Pathological types of oral cancer squamous cell carcinoma and adenoid cystic carcinoma of the majority, and that the degree of malignancy is relatively large, easy to recurrence and metastasis, especially adenoid cystic carcinoma is highly prone to distant metastasis, but also has neurotropic nature. Therefore, we chose five most representative oral cancer cell lines:the squamous cell carcinoma cell line SAS, Tca8113, Buccl885, and adenoid cystic carcinoma cell line ACCM, ACC-2 for screening. DNA binding protein (Inhibitor of DNA binding, Id), are helix-loop-helix protein super-family, relative molecular mass of 13000～20000, which is a group of the lack of basic HLH domain of DNA connecting factor. Id-1 in human so far found four members of the Id proteins (Id-1～4) studied the largest and most important one. The study found:Id-1 in epithelial origin tumors showed high expression, which by inhibiting cell differentiation, promote cell cycle progression and induce cell proliferation and suppress senescence, induced invasion, involved in tumor-mediated angiogenesis and tumor occurrence and development of; in colorectal cancer, esophageal cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer occurrence and development have played an important role.However, in oral cancer, Id-1 expression levels of how their oral cancer cells for growth and invasion capacity of the unknown. This study will detect the expression of Id-1 in this five typical oral cancer cell lines.MethodsOral squamous cell carcinoma cell line SAS, Tca8113, Buccl885, and adenoid cystic carcinoma cell line ACCM, ACC-2 thaw recovery, containing 10% fetal bovine serum in DMEM medium,37℃, the cells containing 5% CO2 incubator closed-culture. Logarithmic growth of cells, using Trizol lysate extracted total RNA, the total RNA reverse transcription of cDNA, and then Id-1 primers for real-time fluorescence quantitative PCR (RFQ-PCR), amplification of the body by adding SYBR Green I, for RFQ-PCR experiment, detection the expression of Id-1mRNA in the five cell lines. And then take on the exponential growth of cells, conventional methods of extrACTINg cells, total protein, first anti-and second anti-incubated for Western Blot test, test Id-1 protein expression in five kinds of cell lines.ResultsExperimental results show that it showed a high Id-1 expression in this five cell lines:squamous cell carcinoma cell line SAS, Tca8113, Buccl885, and adenoid cystic carcinoma cell line ACCM, ACC-2, which, Id-1 expression in ACCM was significantly higher in adenoid cystic carcinoma cell lines,and Id-1 expression in SAS was significantly higher in squamous cell carcinoma cell lines. RFQ-PCR results showed that:Id-1 mRNA expression in ACCM was significantly higher in adenoid cystic carcinoma cell lines(P<0.05), and Id-1 mRNA expression in SAS was significantly higher in squamous cell carcinoma cell lines(P<0.05). Western Blot results showed that:Id-1 protein expression in ACCM was significantly higher in adenoid cystic carcinoma cell lines(P<0.05), and Id-1 protein expression in SAS was significantly higher in squamous cell carcinoma cell lines(P<0.05).ConclusionThese results suggest that, Id-1mRNA and Id-1 protein showed a high expression in this five cell lines:squamous cell carcinoma cell line SAS, Tca8113 Buccl885, and adenoid cystic carcinoma cell line ACCM, ACC-2, which, Id-1 expression in ACCM was significantly higher in adenoid cystic carcinoma cell lines, and Id-1 expression in SAS was significantly higher in squamous cell carcinoma cell lines. We selected two high expression of Id-1 gene in oral cancer cell lines:adenoid cystic carcinoma cell line ACCM and squamous cell carcinoma SAS.Section II The effects of silencing Id-1 gene on the proliferation, invasion and apoptosis in adenoid cystic carcinoma cellsObjectiveAdenoid cystic carcinoma (ACC), occurred in the head and neck, the most common salivary gland malignancy, accounting for 20% of all malignancies, with a high degree of invasion and metastasis characteristics. Because of the understanding of the limitations to the biological nature of ACC, there is not a very effective treatment measures, even if carried out an extensive surgical excision and radiation therapy, most patients will eventually become a recurrence and metastasis. It is urgent to seek treatment for clinically ACC.Id proteins are helix-loop-helix protein super-family, relative molecular mass of 13000～20000, which is a group of the lack of basic HLH domain of DNA connecting factor. bHLH protein dimer formation of DNA with the "E box" (CANNTG) or "N Box" (CACNAG) recognition sequence with a necessary condition, Id proteins with bHLH transcription factors form a heterodimer, interference with DNA, combination of blocking its transcriptional activation of downstream molecules, inhibit gene expression, thus inhibiting normal cell differentiation and promote cell proliferation. Id-1 in human so far found four members of the Id proteins (Id-1～4) studied the largest and most important one. The study found:Id-1 in epithelial origin tumors showed high expression, which by inhibiting cell differentiation, promote cell cycle progression and induce cell proliferation and suppress senescence, induced invasion, involved in tumor-mediated angiogenesis and tumor occurrence and development of; in colorectal cancer, esophageal cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer occurrence and development have played an important role. However, in the ACC, what ahout Id-1 expression levels and how it effects ACC of the growth and invasive ability is unknown.Found in previous studies with tumor-related Id-1 signaling pathway there are many, there is Akt-mediated Wnt signaling pathway, p53 and NF-kB signal-pass, PI3K/Akt/NFKB signaling pathway, ERK/MAPK signaling pathway and so on. Our preliminary study found that expression levels of Id-1 in normal salivary gland tissue is very low (or even difficult to detect), but having a high expression in salivary adenoid cystic carcinoma, indicating the level of Id-1 expression increased normal cells may be breaking the oncogene and tumor suppressor genes balanced relationship of checks and balances, thereby activating one or more of a certain signal transduction pathways to promote cell cycle progression, inhibit cell differentiation, promote cell proliferation, thereby inducing normal cells to tumor cell transformation, to increase through the lymphatic channels and blood Road to the surrounding lymph nodes and distant organs, invasion and metastasis of opportunities.We use a good screening Id-1siRNA sequence, the application lipofectamine 2000 transfection, to silence Id-1 gene in ACCM cell lines, and then test proliferation, invasion and apoptosis changes in ACCM cell.MethodsThe high metastasis adenoid cystic carcinoma cell lines ACCM were contained in DMEM medium containing 10% fetal bovine serum,37℃,5% CO2 incubator in the closed-culture. We use a good screening Id-1siRNA sequence, the application lipofectamine 2000 transfection, to silence Id-1 gene in ACCM cell lines. RFQ-PCR, Western blot and immunofluorescence assay were used to test to confirm the successful disruption. MTT assay was measured at 570 nm optical density (OD), cell growth curve was recorded before and after interference. Transwell chamber was used to test the changes in cell invasiveness before and after the interference. Flow cytometry test the changes in cell apoptosis before and after the interference. Western blot were used to test the protein expression of ki67,c-myc and p21.ResultsThe results showed that RFQ-PCR, Western blot test and immunofluorescence assay to confirm the successful interference. MTT assay recording cell growth curve before and after the interference showed that:after silencing Id-1 gene, the proliferation of ACCM was decreased, and was significantly different with control (0.5806±0.0063 vs 0.7469±0.0424,P<0.01; 0.6378±0.0040 vs 1.0110±0.0010, P<0.01; 0.7679±0.0036 vsl.2774±0.0036, P<0.01). Transwell chamber test results showed that: after silencing Id-1gene, the invasiveness of ACCM was decreased, and was significantly different with control (10.6667±2.0656vs29.8333±1.7224, P<0.01). Flow cytometry showed that:after silencing Id-1 gene, the cell number of apoptosis of ACCM was increased, and was significantly different with control ((23.2767±1.2872) % vs(2.2467±0.0924)%, P<0.01). Western blot test results showed that:after silencing Id-1 gene, and was significantly different with control, the expression of ki67and c-myc were reduction, however, the expression of p21 was upregulation.ConclusionThe results suggested that after RNA interference silencing by Id-1 gene, ACCM growth was inhibited,invasion ability were inhibited, apoptosis increased; after silencing Id-1 gene, the expression of ki67and c-myc were reduction,however, the expression of p21 was upregulation.lts possible mechanism is through silence, Id-1 gene, adenoid cystic carcinoma cell lines showed high expression of ACCM,had Id-1 expression at low or no expression of what might be breaking the cancer gene and tumor suppressor genes balanced relationship of checks and balances returned to normal, the activation of a certain of the signal transduction pathway to be inhibited or shut down, inhibit cell cycle progression and promoting cell differentiation, inhibit cell proliferation, inhibition of blood through the lymphatic channels and the Road to the surrounding lymph nodes and distant organs, invasion and metastasis opportunities. However, the precise signal transduction pathway of Id-1 in ACC to regulate the growth, invasion and of, remains to be further research and discussion.SectionⅢEffects of silencing Id-1 gene on the proliferation, invasion and apoptosis in oral squamous cell carcinoma cellsObjectiveOral squamous cell carcinoma (oral squamous cell carcinoma, OSCC) accounts for more than 90% of oral cancer, in 2005 WHO classification of pathology and genetics head and neck cancer will be oral squamous cell carcinoma is defined as "a different degree of differentiation of invasive tumors, tend to the early, extensive lymph node metastasis, mainly in the 40 to 70 years of age who smoke or alcohol abuse. oral cancer is highly malignant tumor, although the oncologist, the surgeon's continuous efforts in the past 20 years, Village of oral cancer mortality rate declined slightly, but its five-year survival rate was still only 41%～79.5%, which prompted efforts to find the factors associated with tumor prognosis in order to take a more targeted measures for the prevention and treatment.Id proteins are helix-loop-helix protein super-family, relative molecular mass of 13000～20000, which is a group of the lack of basic HLH domain of DNA connecting factor. bHLH protein dimer formation of DNA with the "E box" (CANNTG) or "N Box" (CACNAG) recognition sequence with a necessary condition, Id proteins with bHLH transcription factors form a heterodimer, interference with DNA, combination of blocking its transcriptional activation of downstream molecules, inhibit gene expression, thus inhibiting normal cell differentiation and promote cell proliferation. Id-1 in human so far found four members of the Id proteins (Id-1～4) studied the largest and most important one. The study found:Id-1 in epithelial origin tumors showed high expression, which by inhibiting cell differentiation, promote cell cycle progression and induce cell proliferation and suppress senescence, induced invasion, involved in tumor-mediated angiogenesis and tumor occurrence and development of; in colorectal cancer, esophageal cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer occurrence and development have played an important role. However, in OSCC, Id-1 expression levels of how their growth and invasion of OSCC for the ability of research yet to be confirmed.Found in previous studies with tumor-related Id-1 signaling pathway there are many, there is Akt-mediated Wnt signaling pathway, p53 and NF-kB signal-pass, PI3K/Akt/NFkB signaling pathway, ERK/MAPK signaling pathway and so on. Our preliminary study found that normal salivary gland tissue expression levels of Id-1 is very low (or even difficult to detect), but in salivary adenoid cystic carcinoma is having a high expression, indicating the level of Id-1 expression increased possible to break the normal cell oncogene and tumor suppressor genes within the constraints of a balanced relationship with each other, thereby activating a certain or more signal transduction pathways to promote cell cycle progression, inhibit cell differentiation, promote cell proliferation, thereby inducing normal cells to tumor cell transformation, to increase through the lymphatic channels and blood Road to the surrounding lymph nodes and distant organs, invasion and metastasis of opportunities.We use designed Id-1siRNA sequence, the application lipofectamine 2000 transfection, silence Id-1 gene in OSCC cell lines SAS, and then test proliferation, invasion and apoptosis changes in SAS. Methods The high metastasis adenoid cystic carcinoma cell lines SAS were contained in DMEM medium containing 10% fetal bovine serum,37℃,5% CO2 incubator in the closed-culture. We use a good screening Id-1siRNA sequence, the application lipofectamine 2000 transfection, to silence Id-1 gene in SAS cell lines. RFQ-PCR, Western blot and immunofluorescence assay were used to test to confirm the successful disruption. MTT assay was measured at 570 nm optical density (OD), cell growth curve was recorded before and after interference. Transwell chamber was used to test the changes in cell invasiveness before and after the interference. Flow cytometry test the changes in cell apoptosis before and after the interference.ResultsThe results showed that RFQ-PCR, Western blot test and immunofluorescence assay to confirm the successful interference. MTT assay recording cell growth curve before and after the interference showed that:after silencing Id-1gene, the proliferation of SAS was decreased (0.68055±0.0457vs0.8469±0.0878,P<0.01; 0.73775±0.0866vsl.11098±0.0899,P<0.01;0.8679±0.0442vsl.37743±0.0471,P<0.01). Transwell chamber test results showed that:after silencing Id-1gene, the invasiveness of SAS was decreased (10.1666667±0.012vsl7.8±0.008, P<0.01). Flow cytometry showed that:after silencing Id-1gene, the cell number of apoptosis of SAS was increased ((5.15±3.02)%vs (20.77±2.56)%,P<0.01).ConclusionThe results suggested that after RNA interference silencing by Id-1 gene, SAS growth was inhibited,invasion ability were inhibited, apoptosis increased.Its possible mechanism is through silence, Id-1 gene, oral squamous cell carcinoma cell lines showed high expression of SAS,had Id-1 expression at low or no expression of what might be breaking the cancer gene and tumor suppressor genes balanced relationship of checks and balances returned to normal, the activation of a certain of the signal transduction pathway to be inhibited or shut down,inhibit cell cycle progression and promoting cell differentiation,inhibit cell proliferation,inhibition of blood through the lymphatic channels and the Road to the Surrounding lymph nodes and distant organs, invasion and metastasis opportunities. However, the precise signal transduction pathway of Id-1 in OSCC to regulate the growth,invasion and of, remains to be further research and discussion. 18国家自然基金面上项目(30672339 and 30772269)和SRF for ROCS提供资金资助...