| 【Background】Laryngeal squamous cell carcinoma(LSCC),as the most common cancer of the upper aerodigestive tract,accounts for approximately 14% of head and neck squamous cell carcinoma(HNSCC).The risk factors of LSCC include smoking,alchol drinking,air pollution,papillomavirus infection and etc.Due to the hidden and complex structure of the throat,other types of laryngeal carcinoma are usually difficult to find,causing delay of treatment except the glottic carcinoma.Most of the LSCC have been found in late stages.Recent advances in the multidisciplinary management of the disease,including surgical extirpation or larynx-preservation protocols implementing chemoradiotherapy,but a substantial proportion of patients with localized or advanced disease will relapse and eventually die.Modern studies suggest that tumorigenesis is a complex process with multiple genes and factors involved.Changes in molecular play an important role in the progression of tumors,and which is often preceded by clinical manifestations.Laryngeal carcinogenesis is a complex multistep process involving genetic dysregulation of proto-oncogenes and tumor suppressor genes.To improve the diagnosis and treatment of laryngeal cancer,it is imperative to find effective early diagnosis markers,potential therapeutic targets and available prognostic indexs.Therefore,the research on the molecular mechanisms that underlie LSCC is the basis.MicroRNAs as a class of 17–25 nucleotides small non-coding RNAs,associate with 3’-untranslated regions(3’-UTR)of specific messenger RNAs(mRNAs)leading to transcript degradation or translational inhibition.Increasing evidences demonstrate that dysregulated miRNAs play critical roles in many cell biological processes,including proliferation,invasion,metastasis and apoptosis.MiRNAs play important roles in the development of human malignancies by regulating oncogenes and tumor suppressor genes.The study of the abnormal expression of miRNA in laryngeal carcinoma and its mechanism of the regulation will be helpful to reveal the pathogenesis of human laryngeal carcinoma,and which will provide theoretical basis for improving the diagnosis,treatment and prognosis of cancer by miRNA.Radiotherapy as an important means of tumor therapy has a variety of advantages,but the radioresistance reduces the efficacy in clinical applications,how to improve the radiotherapy of the tumor is also worthy to concern.At present,it has been found that miRNAs are associated with effect of LSCC radiotherapy.The present study shows that miR-24 has the effect of regulating proliferation,differentiation and apoptosis of LSCC cells.Although miR-24 plays essential role in a variety of tumors,the role of miR-24 in LSCC development and its possible molecular mechanism is unclear and remain to be further elucidated.We recruited 15 groups of laryngeal cancer tissue and adjacent tissue to detect miR-24,the results found low expression of miR-24 in LSCC tissues,which supported the feasibility of this study.In this study,we first detected the expression of miR-24 in LSCC cells and tissue samples,and analyzed the correlation between expression level and clinicopathological factors of LSCC.The effect on the biological behavior of LSCC cells by overexpressing of miR-24 were studied.The target gene was predicted and validated.We studied the behavioral changes of LSCC cells after silencing target gene.The effect of miR-24 on radiotherapy sensitivity of LSCC cells and its possible mechanism was also explored.【Aims】1.To detect the expression of miR-24 in human LSCC cells and tissues,and to analyze the correlation between the expression level and the clinicopathological data.2.To study the effect of miR-24 on the proliferation,migration,invasion and apoptosis of LSCC cells.3.To predict the target gene of miR-24 and verify it.4.To study the biological behavior of LSCC cells after silencing the target gene.5.To explore the effect of miR-24 on radiotherapy sensitivity of LSCC cells and its potential mechanism.【Methods】1.The expression of miR-24 in Hep-2 cells,AMC-HN-8 cells and HaCaT cells was detected by qRT-PCR.The expression of miR-24 in tumor tissues and adjacent tissues was also detected by qRT-PCR and the correlation between the expression level and the clinicopathological data was analyzed.2.After constructing Hep-2 and AMC-HN-8 cells with overexpressing of miR-24 by cell transfection technique,the proliferation of Hep-2 and AMC-HN-8 cells were detected by MTT assay and clone assay.Scratch test and Transewell were used to detect the migration and invasion ability,and the effect on the apoptosis was analyzed by flow cytometry assay.3.The target gene of mi R-24 was predicted by bioinformatics software and verified by Dual-luciferase reporter assays.The expression level of target gene protein in miR-24 group and control group was detected by Western blotting.The mRNA and protein of target gene in Hep-2 and AMC-HN-8 cells and LSCC tissues was detected by qRT-PCR and Western blotting,the correlation between mRNA of target gene and mi R-24 in LSCC tissues was analyzed.4.The target gene in Hep-2 and AMC-HN-8 cells was silenced by RNA interference technique,which proliferation and apoptosis were analyzed by MTT,colony formation and flow cytometry assay.5.Hep-2 and AMC-HN-8 cells with high expression of miR-24 were obtained by cell transfection technique,and blank control group and negative control group were set up at the same time.All of the cells were irradiated with 2,4,6 and 8 Gy.The survival fraction of LSCC cells after exposuring to different doses was detected by clonogenic survival assay.Hep-2 and AMC-HN-8 cells with upregulation of miR-24 and control group were irradiated by 6Gy-rays,observing and calculating the number of clones in the two groups.Flow cytometry assays was used to analyze the apoptosis of the two groups after 6Gy irradiation.Western blotting was used to detect the expression of cleaved-caspase-3,cleaved-PARP,total-caspase-3 and total PARP in the two groups after 6Gy irradiation.6.Silencing of XIAP in Hep-2 and AMC-HN-8 cells by RNA interference technique,and setting up the control group.All cells received 2,4,6,8Gy radiation,which survival fraction was observed after different doses of radiation.Silencing XIAP group and control group were treated with 6Gy radiation,the apoptosis of the two groups was observed by flow cytometry assays.Western blotting was used to detect the expression of cleaved-caspase-3,cleaved-PARP,total caspase-3 and total PARP in the two groups after 6Gy irradiation.【Results】1.The miR-24 expression of Hep-2 and AMC-HN-8 cells was lower than that in human keratinocyte cell line.2.The expression of miR-24 in LSCC tissues was significantly downregulated in comparison with that in the adjacent nontumor tissues,and the expression level of miR-24 was significantly correlated with TNM stage and lymphatic metastasis.These results demonstrated that downregulation of miR-24 may play an important role in LSCC development.3.After cell transfection,qRT-PCR assay confirmed the upregulation of miR-24 in Hep-2/miR-24 and AMC-HN-8/miR-24 cells.4.The effects of miR-24 expression on LSCC proliferation were determined by MTT and colony formation assays.It was observed that upregulation of miR-24 significantly reduce proliferation of LSCC cells.Likewise,the capacity of colony formation in Hep-2/miR-24 and AMC-HN-8/mi R-24 cells were significantly reduced,when compared to the control cells.Flow cytometry was performed to the effects of miR-24 expression on apoptosis of LSCC cells,and it was found that overexpression of miR-24 could significantly enhance apoptosis in LSCC cells.These results suggested that miR-24 could inhibit the proliferation,invasion and metastasis for LSCC cells,also increase its apoptosis.5.The target gene of miR-24 was predicted by bioinformatics software.XIAP was selected as the potential target gene.Luciferase activity assay showed that miR-24 significantly inhibited the luciferase activity compared with the negative control miR-NC,but had no effects on the luciferase activity of the reporter vector containing 3’-UTR of XIAP with six point mutations in the miR-24-binding site,suggesting that miR-24 could interact directly with the 3’-UTR of XIAP mRNA.6.Western blotting showed that the expression of XIAP was significantly downregulated in Hep-2 cells with upregulation of miR-24 compared with the negative control group.7.The expression of XIAP in Hep-2 and AMC-HN-8 cells was detected by qRT-PCR and Western blotting,the results showed that the expression of XIAP mRNA and protein in Hep-2 and AMC-HN-8 cells was significantly higher than that in HaCaT cells.It was observed that the expression level of XIAP mRNA and protein in LSCC tissues was significantly higher than that in the adjacent nontumor tissues.It was showed that a statistically significant inverse correlation was observed between XIAP mRNA and miR-24(r =-0.508;P<0.001).Therefore,high expression level of XIAP in LSCC tissues correlated with low level of miR-24 expression.8.XIAP in Hep-2 and AMC-HN-8 cells was silenced,Western blotting showed that the expression level of XIAP in silent group was significantly lower,there was no change in the no silent group and the untreated group.9.MTT and cell colony formation assay showed that,comparing to cells with no silencing XIAP,Hep-2 and AMC-HN-8 cells with silencing XIAP were inhibited,and the number of clones were less.The apoptosis of Hep-2 and AMC-HN-8 cells with silencing XIAP was significantly higher than that of the control group.10.Clonogenic survival assay showed that:(1)The survival fraction of Hep-2 and AMC-HN-8 cells(whether transferred to miR-24 or transferred to miR-NC)which treated by different doses of radiation(2,4,6,8 Gy)were decreased,and in the 0-8Gy range decreased with the dose increased(2)Compared with control group transfected with miR-NC,Hep-2 and AMC-HN-8 cells with high expression of miR-24 were decreased significantly in each dose group,which indicated that radiation could reduce the survival fraction of Hep-2 and AMC-HN-8 cells,the higher expression of miR-24 could induce cell to further die.11.Cell clone formation assay after irradiation showed that:(1)The capacity of colony formation in Hep-2 and AMC-HN-8 cells were significantly reduced after irradiation(6Gy),when compared to the control cells;(2)After irradiation with the same dose(6Gy),the number of clones in Hep-2 and AMC-HN-8 cells with high expression of miR-24 were less than the control group,which indicated that irradiation could inhibit Hep-2 and AMC-HN-8 cells proliferation,cells with high expression of miR-24 which could be further enhanced.12.Analysis of flow cytometry after irradiation showed that:(1)The apoptosis of cells was increased after receiving 6Gy ray irradiation(both of Hep-2 and AMC-HN-8 cells transfected with mi R-24 or miR-NC);(2)The apoptosis of Hep-2 and AMC-HN-8 cells transfected with miR-24 was higher than that of blank control group,which indicated that radiation could induce apoptosis of Hep-2 and AMC-HN-8 cells,high expression of miR-24 could further increase the apoptosis.13.Western blotting analysis found that:(1)Whether or not with high expression of miR-24,the cleaved-caspase-3 and cleaved-PARP in Hep-2 and AMC-HN-8 cells were significantly higher than those in the untreated group,and the totol caspase-3 and total PARP were reduced.(2)The Hep-2 and AMC-HN-8 cells transfected with miR-24 were higher than blank control group,which indicated that radiation could activate caspase-3,high expression of miR-24 further enhanced the activity.miR-24 could enhance the radiosensitivity of LSCC cells by enhancing irradiation-induced caspase-3-dependent apoptosis.14.The same radiotherapy experiments which were performed on Hep-2 and AMC-HN-8 cells with high miR-24 were also used on Hep-2 and AMC-HN-8 cells with silencing XIAP,and the results showed the mimic outcomes were obtained.【Conclusions】1.miR-24 has a lower expression level in LSCC cells and tissues.The expression level of miR-24 in tissues is related to TNM stage and lymphatic metastasis.2.miR-24 can inhibit the proliferation and migration of LSCC cells,and enhance the apoptosis of LSCC cells.3.XIAP is a direct target gene for miR-24.XIAP enhances the proliferation of LSCC cells and inhibits its apoptosis.XIAP is highly expressed in LSCC cells and tissues,and miR-24 in LSCC tissues is inversely correlated with XIAP-m RNA.4.Radiation can reduce the survival fraction of LSCC,inhibit its clonal formation,and enhance its apoptosis.miR-24 can enhance radiosensitivity of LSCC cells by enhancing irradiation-induced caspase-3-dependent apoptosis.Silencing XIAP can enhance the radiosensitivity of LSCC cells as well as upregulation of miR-24.In conclusion,miR-24 can inhibit the proliferation,migration and invasion of LSCC,also promote the apoptosis of LSCC and increase the sensitivity of LSCC cells to irradiation by enhancing irradiation-induced caspase-3-dependent apoptosis,which was very likely to target with XIAP.miR-24 can be used as a potential marker for the diagnosis and prognosis of LSCC.miR-24 in combination with radiotherapy is expected to be an effective way to improve the treatment of LSCC... |
PDF Full Text Request