| BackgroundHypopharyngeal cancer is a type of malignant tumor originating from the hypopharynx.Its pathological type is mainly squamous cell carcinoma,which is closely related to alcohol abuse and smoking.As an upper digestive respiratory tumor,in the surgical treatment of head and neck surgery,hypopharyngeal cancer is the most sophisticated one.In the early stage,hypopharyngeal cancer has no specific symptoms,and once diagnosed,it is already in the stage of tumor progression.Hypopharyngeal cancer is prone to destroy the physiological function of the upper digestive tract.From the histological level,because the hypopharynx lacks the natural biological barrier and the lymphatic drainage in the neck is abundant,it is tend to lead to multiple primary malignant tumor(MTMT)/recurrence.This makes the treatment of hypopharyngeal cancer difficult,and the hypopharyngeal cancer is easy to relapse,so hypopharyngeal cancer is a tumor that is hard to treat in the upper aerodigestive tract.Although the current treatment of hypopharyngeal cancer has formed a comprehensive treatment consensus of surgery followed by radiotherapy and chemotherapy,and the treatment is more abundant than before,the treatment effect of hypopharyngeal cancer is still not optimistic.For now,local recurrence and distant metastasis remain the challenges of hypopharyngeal treatment.Individualized treatment of tumors is the current direction of cancer diagnosis and treatment.Based on their own conditions,designing personalized treatment and follow-up programs for patients is the key to tumor diagnosis and treatment Diagnosing the tumor recurrence in time,can not only greatly improve the effectiveness of treatment,but also improve the prognosis.In addition to traditional TNM staging,many molecular tumor markers have been used in the prognosis of hypopharyngeal cancer.The judgment of the prognosis of hypopharyngeal cancer and the understanding of tumor biology have thus been improved.As a molecular marker,such as genome,the drawback is they are susceptible to physical and chemical factors in the environment.Enzymes,humidity,temperature and many other factors may cause it to degrade or deteriorate,thereby interfering with its expression.For specimens that clinically common stored such as blood products and paraffin sections,it is hatd to detect molecular markers.miRNA is a small RNA synthesized in the human body.The regulation of development and immune function,involvement of various organ diseases and tumor development and outcomes are all involved in miRNA.miRNAs are widely distributed,they can express in tissues,plasma,and body fluids.Because the express of miRNA is quite stable,it can be stably expressed in paraffin tissues for a long time,so miRNA is a very suitable molecular marker.At present,in the related research on the role of miRNA in the prognosis of hypopharyngeal carcinoma,there is a lack of support of large sample data.The aim of this study was combine survival analysis models,to screen for miRNAs which associated with prognosis of hypopharyngeal carcinoma in large sample cases and to evaluate their clinical applicability by high-throughput methods.The biological function of miRNA was verified in vitro cell experiments,and its target genes were searched by prediction,and its regulation mechanism was explored.This provided a new direction for the prevention and treatment of hypopharyngeal carcinoma and molecular biological behavior.Part 1:The profile of miRNAs in hypopharyngeal carcinoma and the correlation between miR-4451 and miR-200a-3p in prognosisObjectivemiRNA expression profiles which are differentially expressed in hypopharyngeal carcinoma tissues and plasma were screened by microarray technique and qPCR.And survival analysis was used to filter prognosis-relating miRNAs which were used to evaluated correlating clinical value eventually.MethodsThe clinical data and epidemiological data of the patients who were diagnosed as hypopharyngeal carcinoma and underwent surgeries between January 2011 and January 2016 were collected.And the patients was divided into the following two groups:Patients from July 2015 to January 2016 were included in the expression verification group to collect tumor tissues,mucosal tissues and plasma samples for detecting and verifying the expression of miRNAs;Patients from January 2011 to June 2015 were included in the survival analysis group,and pathological sections of each were also collected to detecting the expression of miRNAs,then patients were followed up for survival to verify the correlation between miRNAs and prognosis of hypopharyngeal carcinoma.miRNAs in tissues were extracted.After the quality control,the AgilentHuman miRNA microarray(070156)was used for high-throughput screening of candidate miRNAs.The following two screening strategies were used in this study:(1)screening of differentially expressed miRNAs in the hypopharyngeal carcinoma group and mucosa group;(2)Patients were divided into long-term survival group and short-term survival group by median survival time,and the differentially expressed miRNAs in the tumor pathological sections of the two groups were screened by microarray.Candidate miRNAs expression levels were detected by qPCR in 40 pairs of hypopharyngeal carcinoma tumors and mucosal tissues of the expression verification group to confirm differential expression in hypopharyngeal carcinoma.The levels of miRNAs expression in plasma were also tested through qPCR,and its correlation with tissue expression level was studied.Then clinical value was assessed by comparing the difference in plasma miRNAs expression levels between patients and normal volunteers.The miRNAs that were differentially expressed in the hypopharyngeal carcinoma tissues after verification,the expression levels were detected in the tumor sections of the survival analysis group by qPCR.And Log-rank was used to test the difference in survival time between different expression groups through Kaplan-Meier survival curve.Based on the clinical and epidemiological data,the risk ratio of prognosis-related miRNAs and the multi-factor adjusted risk ratio were studied by Cox risk model,and the correlation between them was analyzed.ResultsEleven differentially expressed miRNAs were screened by microarray in the hypopharyngeal carcinoma group and mucosa group.Among them,let-7c-5p,miR-126-3p,miR-195-5p,miR-29c-3p,miR-451a,miR-497-5p were highly expressed in the tumor group,and the low expression were miR-106b-5p,miR-3195,miR-424-5p,miR-4443,miR-93-5p.Through microarray screening in the pathological sections of the long-term survival group and the short-term survival group of the hypopharyngeal carcinoma,high-expression miRNAs including miR-4451,miR-3605-5p,miR-3161,miR-30b-5p,miR-200a-3p,miR-7150,miR-378b,miR-5088-5p were found in the short-term survival group,and low-profile ones,such as miR-200c-3p,miR-429,miR-4688,miR-4701,miR-6808-5p,miR-6813-3p.The above candidate miRNAs were verified by qPCR in 40 pairs of hypopharyngeal carcinoma and mucosal tissues,and 9 miRNAs were differentially expressed.In the hypopharyngeal carcinoma,miR-126-3p(FC=0.55),miR-195-5p(FC=0.0.71),miR-29c-3p(FC=0.63),miR-451a(FC=0.28),miR-497-5p(FC=0.49)was low expression,miR-106b-5p(FC=1.67),miR-93-5p(FC=1.85),miR-4451(FC=1.91)and miR-200a-3p(FC=1.36)were high expression.Among the above differentially expressed miRNAs,only the plasma expression level of miR-93-5p was correlated with tissue expression,and the Spearman correlation coefficient was 0.516.The ROC test showed an AUG of 0.701(p=0.002),a sensitivity of 0.625 at a cut-off value of 0.143,a specificity of 0.628,and an approximated index of 0.997.This study also confirmed that RNU48 is a suitable internal reference gene for hypopharyngeal carcinoma-related experiments.qPCR was used to detect the differentially expressed miRNAs in 372 cases of hypopharyngeal carcinoma tumors in the survival analysis group.Log-rank test showed that the expression levels of miR-4451 and miR-200a-3p were associated with prognosis.The expected survival time of miR-200a-3p in low expression group was 64.8 months(95%CI:59.4-70.1),and in high expression group was 52.8 months(95%Cl:46.8-58.7).And The expected survival of the miR-4451 in low expression group was 65.6 months(95%CI:30.2-71.0),and the high expression group had a survival of 52.2 months(95%CI:46.3-58.1)Statistical differences of all mentioned above were significant.The prognosis of the miR-200a-3p and miR-4451 high expression groups was poor.The one-way Cox proportional hazard model showed that miR-200a-3p in high expression group had a hazard ratio of 1.51(95%CI:1.09-2.09)relative to the low expression group,and miR-4451 was 1.64(95%CI:1.18-2.28).Multi-factor Cox analysis was performed with T stage,N stage,pathological grade and primary site as covariates.The above risk ratios were adjusted to 1.42(95%CI:1.02-1.98)and 1.47(95%Cl:1.06-2.06)respectively.The difference was statistically significant.The proportion of high expression of miR-200a-3p and miR-4451 significantly increased in T4 patients,and the high expression ratio of miR-200a-3p increased in N2 and N3 patients.The prognostic index constructed by miR-4451 and miR-200a-3p expression levels showed that both high expression led to risk ratios increased to 1.67(single factor,95%CI:1.19-2.33),1.56(multifactor,95%CI:1.11-2.20).Harrel's C-index showed an increasing ability in the prediction of the Cox model with the addition of miR-4451 and miR-200a-3p.ConclusionsIn hypopharyngeal cancer tissues,miR-126-3p,miR-195-5p,miR-29c-3p,miR-451a,miR-497-5p were low-expressed,while miR-106b-5p,miR-93-5p,miR-4451 ?miR-200a-3p were high-expressed.The finding of high expression of miR-93-5p in patients' plasma indicates that it could be used as a diagnostic index.In addition,high expression of miR-200a-3p and miR-4451 are associated with poor prognosis,which provides a new direction in molecular diagnosis of hypopharyngeal cancer prognosis.Part 2:miR-4451 regulates lipid metabolism and biological behavior in FaDu cell line by targeting ADHD5ObjectiveThe effects of miR-4451 and miR-200a-3p screened in the part one on FaDu cell line were verified by experiments in vitro,and the downstream target genes were further searched for functional verification.And the effects of miRNA-regulated target genes on the biological behavior of hypopharyngeal carcinoma were studied at both molecular and cellular levels.MethodsUsing FaDu cell line as a research object,and the levels of candidate miRNAs in it were interfered by transfecting miRNA mimics and inhibitors.Through CCK8 experiment,Transwell migration and invasion experiments,the effects of different miRNA expression levels on proliferation,migration and invasion of hypopharyngeal carcinoma FaDu cells were observed.TargetScan database,miRWalk database and mRNA chip were used to screenpotential miRNA target gene.And further narrowing predictive range of target gene was done through Starbase.The target gene was screened and identified by transfecting miRNA mimics to interfere with the expression level of miRNA in cells,and then detecting the expression of candidate target gene at mRNA levels through qPCR.The 3'UTR plasmid of pmirGLO target gene was constructed,which was used to co-transfect with mimics in Fadu cells,and a dual luciferase reporter gene assay was performed to verify the target gene.The transcription levels of the selected target genes were compared by qPCR in tumor and mucosal tissues of 40 pairs of hypopharyngeal carcinomas in the expression verification group.Next,tumor sections of 57 patients in the survival analysis group underwent immunohistochemistry staining,semi-quantitatively tested in protein expression levels,and their survival data were used to study the correlation with the prognosis of hypopharyngeal carcinoma.siRNAs of the target genes were transfected into FaDu cells to knock down its expression,followed by CCK8 assay,Transwell migration and invasion assay to observe the effects of target genes interference on proliferation,migration and invasion of hypopharyngeal carcinoma FaDu cells.A related recovery experiment was carried out for testing the function of miRNAs,mainly by co-transfection of miRNA inhibitors and siRNAs of target genes to study the effects of the interaction on Fadu cell migration and lipid metabolism.ResultsABHD5(NM 016006),SLC37A2(NM 001145290)and ZNF527(NM 032453)were screened as potential target genes by biomarker database and miRNA microarray.According to transfecting miR-4451 mimics and other experiments mentioned above,it was found that low expression of ABHD5 at both mRNA and protein levels.Through dual luciferase reporter gene experiments,it was found that co-transfection of pmirGLO-ABHD wild type and miR-4451 mimics significantly decreased FL/RL,confirming that ABHD5 is a target gene of miR-4451.In the 40 pairs of hypopharyngeal carcinoma and mucosal tissues of the validation group,ABHD5 was differentially expressed at the mRNA level.Immunohistochemistry analysis showed that among the 57 patients with hypopharyngeal cancer between 2012 and 2013 who with low expression of ABHD5 had a poor prognosis.Transfection of AHBD5 siRNAs promoted the proliferation,migration and invasion in FaDu cells.miRNA functional recovery experiments showed that transfection of ABHD5 restored the effects of miR-4451 on cell proliferation and lipid metabolism.Low expression of ABHD5 and high expression of miR-4451 could increase the lipid droplet content of tumor cells and promoted the migration of FaDu cells.ConclusionsmiR-4451 promotes proliferation,migration and invasion in hypopharyngeal cancer FaDu cells.miR-4451 affects lipid metabolism and biological behavior of FaDu cells by targeting regulating ABHD5.And the low expression level of ABHD5 in hypopharyngeal carcinoma could be a new prognostic factor.ABHD5 can promote the proliferation,migration and invasion of hypopharyngeal cancer FaDu cells.This part of the study provides a new direction for understanding the biological behavior and lipid metabolism of hypopharyngeal carcinoma. |
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