Expression And Functions Of Circular RNA MART3 In Hypopharyngeal Squamous Cell Carcinoma

BackgroundHypopharyngeal squamous cell carcinoma(HSCC)is one of the most malignant head and neck squamous cell carcinoma,accounting for 5%-15%of head and neck squamous cell carcinoma.It occurs mainly in the piriform sinus,posterior hypopharyngeal wall and posterior annular area of the hypopharynx,which occurs in the hypopharyngeal piriform fossa most frequently.Although the incidence of hypopharyngeal carcinoma is relatively low,most of the patients were diagnosed at the advanced stage due to the insidious early symptoms.Therefore,lymph node metastasis occurs in most of patients diagnosed with hypopharyngeal carcinoma,and some patients even have cervical lymph node enlargement as the first symptom.In present,due to the recent improvements in modern comprehensive treatment strategies including surgery,chemotherapy and radiotherapy,and the 5-year survival rate of these patients is about 35%,and HSCC patients are also vulnerable to relapse.So it is particularly important to find new tumor diagnostic markers and therapeutic targets to diagnose hypopharyngeal cancer early and improve the clinical outcomes of patients.In recent years,circular RNA(circRNA),as a special type of noncoding RNA(ncRNA)with a closed circular structure,which is different from traditional linear RNA,and it exists in lots of eukaryotic transcriptome.Currently,circRNA is a hot topic in the field of RNA research.Most circRNAs are generated from exons,and circRNAs are highly conserved in different species,and specific in tissues and developmental stages of different species.Due to its highly conserved circular structure and insensitivity to ribozyme,circRNAs are more stable than linear RNAs,which endows circRNAs having obvious advantages in the prospect of development and application as new clinical diagnostic markers and therapeutic targets.In addition,circRNAs can serve as competitive endogenous RNAs(ceRNA)in cells and bind miRNAs competitively,through which they can regulate the expression of target genes.In recent years,more and more experimental evidence has shown that circRNAs can regulate transcription,splicing and gene expression processes by acting as miRNA sponges,splicing or transcriptional regulators and RNA-binding proteins.Nowadays,a large number of circRNAs have been proved to play an important role in the development and progression of a variety of human tumors.CircRNAs are related with the regulation of multiple biological functions including proliferation,metastasis,invasion,apoptosis and drug resistance of various tumorous cells,including liver cancer,gastrnc cancer,colon cancer and bladder cancer.Different circRNAs were expressed differently in various tumors,suggesting that circRNA could play a crucial role in promoting and inhibiting cancer.Therefore,circRNA,as a new tumor diagnostic marker and potential therapeutic target,has an important clinical application prospect.At present,the biological functions of circRNAs in hypopharyngeal cancer have rarely been reported.This study was divided into the following two parts:(1)the second-generation high-throughput circRNA sequencing technology was used to detect the difference in circRNA expression between HSCC tissues and adjacent normal tissues.The results showed that,compared with adjacent normal tissues,a vaiety of circRNAs were up-regulated in HSCC tissues.Therefore,we use quantitative real time polymerase chain reaction(qRT-PCR)method to verify several differentially expressed circRNAs in HSCC tissue.And through the preliminary experiments,we confirmed that circMART3 may play an important role in HSCC development,so we performed qRT-PCR to detect the relative expression level of circMART3 in 50 pairs of HSCC tissues and adjacent normal tissues.The correlation between the expression level of circMART3 and the clinicopathological characteristics of HSCC patients was analyzed,and we also evaluated the relationship between the expression level of circMART3 and the clinical prognosis of patients.(2)We utilized the small interfering RNA(siRNA)technology to knock down the expression of circMART3 in vitro cell model,and we performed cell proliferation,apoptosis,cell cycle and transwell experiments to study the effect of circMART3 on HSCC biological behaviorPart I Expression level of circRNA-MART3 in hypopharyngeal carcinoma and its clinical significanceObjectiveTo screen the difference of circRNAs expression between HSCC tissues and adjacent normal tissues,to verify the differentially expression of circRNAs in HSCC tissues,to detect the relative expression level of circMART3 in 50 pairs of HSCC tissues and adjacent normal tissues,and for analyzing the correlation between the expression level of circMART3 and the clinical characteristics and prognosis of HSCC patientsMethodWe selected 55 patients diagnosed as HSCC by pathological biopsy from January 2011 to January 2014 in the department of otolaryngology,head and neck surgery of Qilu hospital(Shandong university)as the study subjects,and we collected the HSCC tissues and adjacent normal tissues from 55 patients during the surgery.CircRNA expression profiling in HSCC and adj acent normal tissues was detected by using circRNA-seq high-throughput sequencing in 5 pairs of samples,and the expression differences between the two groups were analyzed;we extract RNAs from another 50 pairs of HSCC tissues and adjacent normal tissues to verify the high-throughput sequencing results by using qRT-PCR method.The role of circMART3 in promoting cell proliferation of HSCC cell was determined by preliminary experiments,and then the correlation between circMART3 expression level and the clinicopathological characteristics and prognosis of HSCC patients was analyzed.Among them,Fisher's exact test or chi-square test were used to evaluate the circMART3 expression level and the clinicopathological characteristics of patients,t test was used to compare the differences between the two experimental groups,Kaplan-Meier method was used to draw the survival curve of HSCC patients with high or low circMART3 expression,and log-rank method was used to compare the survival rate of patients in the two groupsResults(1).Compared with adj acent normal tissue mucosa,38 circRNAs were significantly up-expressed in HSCC tissues(P<0.05,fold change>2),and 117 circRNAs were significantly decreased in the HSCC tissues(P<0.05,fold change>2).(2).We selected two up-regulated circRNAs(circMART3,circPTBP3)and two down-regulated circRNAs(circPHF21A,circRSF1)in HSCC tissues,and qRT-PCR method was used to verify the high-throughput sequencing results in 20 pairs of HSCC tissues and adj acent normal tissues.QRT-PCR results showed that the relative expression levels of circMART3 and circPTBP3 were consistent with the results of high-throughput sequencing,and the pre-experiment of HSCC cell proliferation showed that circMART3 play a role in promoting the proliferation of HSCC cells,while circPTBP3 have not the corresponding biological function(3).QRT-PCR results of 50 pairs of HSCC samples showed that the relative expression of circMART3 in HSCC tissues was(1.4957± 0.9913),which was significantly higher than the relative expression of circMART3 in adjacent normal tissues(1.0000± 0.7325,P = 0.003 7).HSCC patients with high circMART3 expression had worse T staging(P = 0.022),earlier lymph node metastasis(P =0.038),and later clinical staging(P = 0.026).In addition,based on the median relative expression level of circMART3,we separated patients with hypopharyngeal cancer into low expression group and high expression group,and we found that the overall survival of patients with high circMART3 expression group was significantly lower than that of patients with low circMART3 expression group(P = 0.015).Conclusion(1).CircRNAs have important biological effects in the development of HSCC,and are expected to become new diagnostic markers and therapeutic targets for HSCC.(2).We firstly found that circMART3 is up-expressed in HSCC,and the expression level of circMART3 was significantly associated with T staging,lymph node metastasis,clinical stage,and overall survival in patients with hypopharyngeal cancer.Therefore,circMART3 may play important biological functions in the occurrence and development of HSCC,suggesting that it can be used as a new biological indicator to evaluate the clinical prognosis of HSCC.Part II Verification of circMART3 and its effect on the biological functions of HSCC cellsObjectiveTo verify the circular structure and characteristics of circMART3,and to study the effects of circMART3 on the biological behaviors of HSCC cells,such as cell proliferation,cell cycle,apoptosis,migration and invasion.MethodThe cDNA was synthesized from RNA that extracted from the FaDu cells,we performed PCR by utilizing divergent primer.The stability of circMART3 was verified by enzyme tolerance assay.SiRNA was transfected into HSCC cells(FaDu cells)to knock down the expression level of circMART3.The relative expression of circMART3 in FaDu cells was detected by qRT-PCR,and the silencing efficiency was evaluated.CCK8 assay was used to detect the effect of circMART3 on the proliferation of FaDu cells,Transwell assay was used to detect the effect of circMART3 on the migration and invasion of FaDu cells,and flow cytometry was used to detect the effect of circMART3 on the cell cycle and apoptosis of FaDu cells.ResultsSanger sequencing results showed that circMART3 splicing site and its sequence were correct,which is consistent with the characteristics of circular structure.Enzyme tolerance experiments showed that circMART3,as a circma,had stable molecular structure and was able to resist with the digestion of Ribonuclease R(RNase R).The results of QRT-PCR showed that the expression level of circMART3 in FaDu cells(si-circMART3)after siRNA interference was significantly lower than that in control group cells(si-NC).CCK8 assay results showed that the proliferation of FaDu cells with low circMART3 expression was significantly inhibited compared with the control group cells(P<0.05).The results of Transwell assay showed that knocking down the expression level of circMART3 could significantly reduce the migration and invasion ability of FaDu cells.Flow cytometry was used to detect the effect of circMART3 on the cell cycle and apoptosis of FaDu cells,and the results showed that decreased circMART3 expression had no significant effect on every cell cycle of FaDu cells(P>0.05),but the apoptosis rate of FaDu cells with lower circMART3 expression was significantly higher than that in control group cells(P<0.05).ConclusionAs a circRNA,circMART3 owns the characteristics of circular structure and high stability.CircMART3 can promote the proliferation of HSCC cells,inhibit cell apoptosis,and improve the migration and invasion of HSCC cells.