| Objective: Laryngeal squamous cell cancer is a common malignant cancer with a high incidence of dying in head and neck diseases.The treatment of laryngeal cancer includes sugery,chemotherapy,and radiotherapy.However,these different treatments have their own advantage and disadvantage.Because of poor survival of patients,the effect of these therapy treatment is still not ideal.Therefore,searching for the possible therapeutic biomarkers has become a hotspot of the oncology research.Studies have shown that long non-coding RNA(lnc RNA)plays a key role in tumorigenesis,but the molecular regulatory mechanism and corresponding clinical significance of lnc RNA in tumos is still unclear.Lnc RNA NEAT1 located in the nucleus,and it has an important role in occurrence and progrsioon of cancers.Studies have demonstrated that NEAT1 could regulate the expression of gene by regulating and stabilizing m RNA.However,the related mechanism of NEAT1 in laryngeal cancer has not been fully explored,which attracted our attention.Micro RNA(miRNA)is a class of single-stranded,non-coding RNA with a length of 18-25 nucleotides.Mi RNA could inhibit m RNA synthesis or degrade m RNA directly by binding to their target m RNA molecule.Then,at the posttranscriptional level,miRNA may participate in gene regulation.Now,many studies have demonstrated that lnc RNA can interact with miRNA,especially the competitive mechanism of endogenous RNA(ce RNA).This means lnc RNA could bind to some specific miRNA competitively as a ce RNA and reduce the regulation of miRNA on the downstream target gene.The purpose of this study was to explore the molecular regulatory mechanism: NEAT1 might regulate the expression of 3-phosphoinositidedependent protein kinase-1(PDK1)by competitively binding miR-139-5p,and it influence the biological behavior of laryngeal squamous cell cancer.Methods:Part I: In this study,the expression,clinicopathological factors and patients’ prognosis of NEAT1 in head and neck squamous cell cancer samples were analyzed by UALCAN website.Meta-analysis was also performed to analyze the correlation between clinicopathological factors and prognosis of patients and NEAT1 overexpression.From Liaoning cancer hospitital & institute between November 2019 and July 2011,30 laryngeal squamous cancer patients were collected.The expression of NEAT1 in tumor tissues and ajacent normal tissues cell were detected by real-time quantitative polymerase chain reaction(q RT-PCR).Based on the detection results,NEAT1 was divide into high expression group(17 patients)and low expression group(13 patients).The clinicopathogical data of these patients also collected.Then,the relationship between the expression of NEAT1 and the clinicopathological factors were analyzed.We tested NEAT1 expression in different cells(NP69,AMC-HN-8,TU212,TU177,TU686,and Hep2)by q RT-PCR.AMC-HN-8 and TU212 cells were transfected with control,sh-NC and sh-NEAT1.CCK-8 and clone formation assay were used to detect cell proliferation of AMC-HN-8 and TU212 cells in each group.Wound healing and Transwell test were used to detect migration and invasion of AMC-HN-8 and TU212 cells in each group.Flow cytometry was used to tect the cell cycle and the apoptosis rate of AMC-HN-8 and TU212 cells in each group.Establish a subcutaneous xenograft model of laryngeal squamous cell cancer in nude mice.Nude mice were divided into control,sh-NC and shNEAT1 group.We observed the growth of subcutaneous xenografts in nude mice and measured the volume of tumors.Then,we plotted the growth curve of the tumors.After 4weeks,nude mice were sacrificed,the tumors were removed and weighed.Part II: The potential binding sites between NEAT1 and miR-139-5p were predicted by star Base,miRcode,and LNCediting bioinformatics software.Dual luciferase reporter assay and and RNA immunoprecipitation(RIP)experiment demonstrated the targeted regulation of NEAT1 and miR-139-5p.We tested the miR-139-5p expression in cancer tissues and cells(AMC-HN-8 and TU212)by q RT-PCR.The Pearson method was performed to analyze the relationship between NEAT1 and miR-139-5p expression in cancer tissues.AMC-HN-8 and TU212 cells were transfected with sh-NC,sh-NEAT1,pc DNA-NC,and pc DNA-NEAT1,respectively,then q RT-PCR was performed to detect the effect of up-regulating or down-regulationg NEAT1 on miR-139-5p expression.AMC-HN-8 and TU212 cells were transfected with control,mimics-NC,miR-139-5p mimics,respectively,then CCK-8 assay,Transwell test,and flow cytometry were used to detect cell proliferation,invasion,cell cycle and the apoptosis rate in each group.AMCHN-8 and TU212 cells were transfected with sh-NC、sh-NEAT1、sh-NEAT1+miR-139-5p inhibitor 和 sh-NEAT1+inhibitor-NC,respectively,then CCK-8 assay,Transwell test,and flow cytometry were used to detect cell proliferation,invasion,cell cycle and the apoptosis rate in each group.Establish a subcutaneous xenograft model of laryngeal squamous cell cancer in nude mice.Nude mice were divided into control,agomiR-NC group and agomiR-139-5p group.We observed the growth of subcutaneous xenografts in nude mice and measured the volume of tumors.Then,we plotted the growth curve of the tumors.After 4 weeks,nude mice were sacrificed,the tumors were removed and weighed.Part III: The potential binding site between miR-139-5p and PDK1 was predicted by star Base bioinformatics software.Dual luciferase reporter assay and and RIP experiment demonstrated the targeted regulation of PDK1 and miR-139-5p.We tested the PDK1 m RNA and protein expression in cancer tissues and cells(AMC-HN-8 and TU212)by q RT-PCR and Western blot.30 specimens of tumor tissues and ajacent normal tissues were selected for analysis of PDK1 expression by immunohistochemical staining.The Pearson method was performed to analyze the relationship between PDK1,NEAT1 and miR-139-5p expression in cancer tissues.AMC-HN-8 and TU212 cells were transfected with control,mimics-NC,miR-139-5p mimics,inhibitor-NC,and miR-139-5p inhibitor,respectively,then q RT-PCR and Western blot were used to detect PDK1 expression.AMC-HN-8 and TU212 cells were transfected with control,sh-NC,and sh-NEAT1,respectively,then q RT-PCR and Western blot were used to detect PDK1 expression.AMC-HN-8 and TU212 cells were transfected with pc DNA-NC,pc DNA-NEAT1,miR-139-5p mimics,pc DNA-NEAT1+miR-139-5p mimics,pc DNA-NC+mimics-NC,respectively,then q RT-PCR and Western blot were used to detect PDK1 expression.AMC-HN-8 and TU212 cells were transfected with control,sh-NC,and sh-PDK1,respectively,then CCK-8 assay,Transwell test,and flow cytometry were used to detect cell proliferation,invasion,cell cycle and the apoptosis rate in each group.Establish a subcutaneous xenograft model of laryngeal squamous cell cancer in nude mice.Nude mice were divided into control,sh-NC,and sh-PDK1 group.We observed the growth of subcutaneous xenografts in nude mice and measured the volume of tumors.Then,we plotted the growth curve of the tumors.After 4 weeks,nude mice were sacrificed,the tumors were removed and weighed.Results:Part I: Bioinformatics analysis showed that NEAT1 gene was overexpression in head and neck squamous cell cancer tissue,and the patients with NEAT1 overexpression had higher TNM stage,and lower rate of survival.Meta-analysis showed that high overexpression of NEAT1 had a significantly relationship with tumor size,TNM stage,T stage,lymph node metastasis and distant metastasis(p<0.05).The expression of NEAT1 in cancer tissues was significantly higher than that in adjacent normal tissue(p<0.05).The NEAT1 overexpression was associated with smoking history,tumor size,T stage,TNM stage,and lymph node metastasis(p<0.05).The NEAT1 expression in AMC-HN-8and TU212 cells was significantly increased compared with NP-69 cell(p<0.05),and these two cells were choosed in subsequent experiments.Compared with control and shNC groups,NEAT1 expression was significantly decreased in sh-NEAT1 group(p<0.05).Compared with control and sh-NC groups,the proliferation of cancer cells was significantly decresased(p<0.05).The number of cancer cells migration and invasion was significantly reduced(p<0.05).The proportion of cancer cells in G0/G1 phase were significantly increased,while the rate of cells in S phase were significantly decreased(p<0.05),and the rate of apoptotic of cancer cells was significantly increased(p<0.05).The subcutaneous xenografts of sh-NEAT1 group grew slowly,and the volume and weight of tumors significantly decrease(p<0.05).Part II: The dual luciferase reporter assay demonstrated that the luciferase activity of miR-139-5p mimics group with NEAT1-WT was significantly lower than that of mimics-NC group with NEAT1-WT(p<0.05).However,co-transfection with NEAT1-MUT,the luciferase activity of miR-139-5p mimics groups was not significantly different from that of mimics-NC(p>0.05).RIP experiment showed that NEAT1 and miR-139-5p were significantly enriched in AGO2 group compared with the Ig G group(p<0.05).The expression of miR-139-5p in cancer tissues,AMC-HN-8,and TU212 cells was significantly decreased compared with NP-69 cell(p<0.05).Pearson analysis found that the expression of NEAT1 was negatively correlated with miR-139-5p(p<0.05).Compared with pc DNA-NC group,the expression of miR-139-5p in AMC-HN-8 and TU212 cells in pc DNA-NEAT1 group was significantly decreased(p<0.05);while the expression of miR-139-5p in sh-NEAT1 group was significantly increased compared with sh-NC group(p<0.05).Compared with control and mimics-NC groups,miR-139-5p mimics group was found that the cell proliferation and invasion were significantly reduced(p<0.05);the proportion of cancer cells in G0/G1 phase were significantly increased;while the rate of cells in S phase were significantly decreased(p<0.05),and the rate of apoptotic of cancer cells was significantly increased(p<0.05).Compared with sh-NEAT1+inhibitor-NC group,sh-NEAT1+miR-139-5p inhibitor group was found that the the cell proliferation and invasion were significantly increased(p<0.05);the proportion of cancer cells in G0/G1 phase were significantly decreased,while the rate of cells in S phase were significantly increased(p<0.05),and the rate of apoptotic of cancer cells was significantly decreased(p<0.05).The subcutaneous xenografts of agomiR-139-5p group grew slowly,and the volume and weight of tumors significantly decrease(p<0.05).Part III: The dual luciferase reporter assay demonstrated that the luciferase activity of miR-139-5p mimics+PDK1-WT was significantly lower than that of mimics-NC+PDK1-WT(p<0.05).However,co-transfection with PDK1-MUT,the luciferase activity of miR-139-5p mimics groups was not significantly different from that of mimics-NC(p>0.05).RIP experiment showed that PDK1 and miR-139-5p were significantly enriched in AGO2 group compared with the Ig G group(p<0.05).The expression of PDK1 m RNA and protein in cancer tissues,AMC-HN-8,and TU212 cells was significantly increased compared with NP-69 cell(p<0.05).Immunohistochemical staining results found that PDK1 was mainly expression in cytoplasm and cell membrane in cancer cells,and the expression of PDK1 was significantly higher in tumor tissues(p<0.05).Pearson analysis found that the expression of PDK1 was negatively correlated with miR-139-5p(p<0.05),while it was postively correlated with NEAT1(p<0.05).Compared with control group,the expression of PDK1 in AMC-HN-8 and TU212 cells in miR-139-5p mimics group was significantly decreased(p<0.05);while the expression of PDK1 in miR-139-5p inhibitor group was significantly increased(p<0.05).Compared with control and sh-NC groups,the expression of PDK1 in sh-NEAT1 group was significantly decreased(p<0.05).Compared with other groups,the expression of PDK1 in pc DNANEAT1+miR-139-5p mimics group was significantly decreased(p<0.05);while the expression of PDK1 in sh-NEAT1+miR-139-5p inhibitor group was significantly increased(p<0.05).Compared with control and sh-NC groups,sh-PDK1 group was found that the cell proliferation and invasion were significantly reduced(p<0.05);the proportion of cancer cells in G0/G1 phase were significantly increased(p<0.05),and the rate of apoptotic of cancer cells was significantly increased(p<0.05).The subcutaneous xenografts of sh-PDK1 group grew slowly,and the volume and weight of tumors significantly decrease(p<0.05).Conclusion:1.The expression of NEAT1 is increased in cancer tissues and cells.The expression of NEAT1 was higher in patients with smoking history,T stage,lymph node metastasis and high TNM stage.2.Silencing of NEAT1 can inhibit the proliferation,migration,and invasion of cancer cells.It also can affect the cell cycle and promote the apoptosis of cancer cells.Silencing of NEAT1 could inhibit the growth of laryngeal tumors in nude mice.3.The expression of miR-139-5p is decreased in cancer tissues and cells.Overexpression of miR-139-5p can inhibit the proliferation and invasion of cancer cells.It also can affect the cell cycle and promote the apoptosis of cancer cells. Overexpression of miR-139-5p could inhibit the growth of laryngeal tumors in nude mice.4.The expression of PDK1 is increased in cancer tissues and cells.Silencing of PDK1 can inhibit the proliferation and invasion of cancer cells.It also can affect the cell cycle and promote the apoptosis of cancer cells.Silencing of PDK1 could inhibit the growth of laryngeal tumors in nude mice.5.NEAT1 targeted negative regulation of miR-139-5p expression,miR-139-5p targeted negative regulation of PDK1 expression6.Lnc RNA NEAT1 regulates the expression of miR-139-5p/PDK1 signaling pathway,which affects the biological function of laryngeal squamous cell carcinoma,thereby promotes the progression of laryngeal cancer. |
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