The Expression And Mechanism Of Long-noncoding RNA SNHG16 In The Esophageal Squmous Cell Carcinoma

Background:Oesophageal cancer(EC)is one of the most common eight malignant tumors in the world,with the 6th highest mortality rate.The pathological types of the EC mainly include the squamous cell carcinoma(ESCC)and adenocarcinoma(EAC).Approximately 90% of EC in China are oesophageal squamous cell carcinoma(ESCC),which shows aetiological and pathological characteristics distinct from oesophageal adenocarcinoma(EAC).Although the development of multimodality therapy improved the ESCC patients prognosis,the 5-year overall survival(OS)rate still remains unsatisfactory,account of approximately 15%-25%.It is believed that late diagnosis and tumour metastasis propensity are associated with patients’ poor outcomes.Genetic susceptibility,environmental factors and gene–environment interactions dominantly contribute to the development and progression of ESCC.An in-depth study of the molecular mechanisms of ESCC carcinogenesis and screening specific biomarkers are of particular significance for the ESCC therapy and early diagnosis.The ENCYCLOPEDIA of DNA elements(EN-Code)project has confirmed that 80% of genes in the genome can be transcribed,but less than 2% of genes can be expressed as proteins.RNA molecules that do not encode proteins are called noncoding RNAs(nc RNA)and are divided into micro RNA and lncRNA.Lnc RNAs,a recognized class of noncoding RNAs(nc RNAs)with lengths longer than 200 nucleotides(nt),accounting for 0.03%-0.2% of the total RNA,have limited or no protein-coding capacity.Early genomics studies believed that Nc RNAs except r RNAand t RNA were byproducts of RNA polymerase II transcription and had no biological functions,so they were considered as transcriptional noise.Compared with mRNA,lncRNAs lack of open reading frame(ORF),short nucleotide sequence,fewer exons,and poor conserved primary sequence.Lnc RNAs express in cytoplasm and nucleus,and participate various biological functions at transcriptional and post-transcriptional levels,including chromatin modification,transcription,posttranscriptional regulation and nuclear transport.The abnormal expression of lncRNAs has vital value in the diagnosis and prognosis of disease.As lncRNAs showed characters of more tissue specific or cell-type specific than protein-coding genes,they only expressed at specific developmental stage of diseases,tissues or cells,and displayed distinct biological roles in physiological and pathological settings especially cancers.Previous studies showed that abnormal lncRNAs exist in ESCC,but its specific mechanisms still unclear.Finding specific and sensitive lncRNAs is of great significance for the early detection of ESCC.Part Ⅰ.The aberrantly expressed lncRNAs of ESCC and the expression of SNHG16 in cancerous and precancerous tissuesAims:To explore the expression profile of lncRNA in ESCC,try to find specific lncRNA in ESCC and its precancerous lesions.Methods:1.Esophageal intraepithelial neoplasia(IEN)tissues,cancerous tissues,and paired normal esophagus tissues were obtained from patient those who received endoscopic submucosal dissection(ESD)or oesophagectomy with no chemoradiotherapy previously in the Department of Gastroenterology and Department of Cardiothoracic Surgery at Zhongda Hospital Affiliated of Southeast University from February 2019 to November 2021.Four pairs of ESCC tissues and normal oesophagus tissues were used to obtain a genome-sequence screen.2.SNHG16 expression in primary ESCC tissues,precancerous tissues and matched normal oesophageal tissues were detected by q RT-PCR.The correlation between SNHG16 expression and detailed clinical baseline characteristics of the patients were analyzed.SNHG16 expressions in diverse human ESCC cell lines were detected by q RT-PCR.Results:1.RNA sequences detected 2,145 different lncRNAs,including 175 upregulated and 134 downregulated lncRNAs.Among all the differentially expressed lncRNAs,SNHG16 was one of the most upregulated lncRNAs in ESCC tissues.We focus on SNHG16,for which no function in ESCC has been ascribed since our research begins.2.Public database analysis showed that SNHG16 was highly expressed in a variety of tumors,especially in esophageal cancer,malignant melanoma and testicular tumors.We further examined the expression level of SNHG16 in 25 primary ESCC tissues and matched normal oesophageal tissues and found that SNHG16 was significantly upexpressed in the cancerous tissues.Similarly,we found that SNHG16 expression was high in precancerous samples.The expression level of SNHG16 was correlated with tumour differentiation(P<0.05),suggesting that SNHG16 upregulation might was an early event in the development of ESCC.The level of SNHG16 was increased in ESCC cell lines compared to the normal oesophageal cell line(HET-1A).KYSE410 and KYSE30 were up-regulated 4.9 times and 3.1 times(P<0.05),respectively.Eca109 was up-regulated by 1.5 times,and KYSE140 was up-regulated by 1.4 times(P<0.05).Conclusions:Differentially expressed lncRNAs were found in ESCC,including 175 up-regulated lncRNAs and 134 down-regulated lncRNAs.SNHG16 is highly expressed in ESCC and its precancerous tissues.The expression level of SNHG16 is correlated with the tumor cell differentiation.Part Ⅱ.The in vitro and in vivo effects of SNHG16 on ESCCAims:To investigate the effect of SNHG16 on ESCC cells in vitro and in vivo.Methods:1.The overexpressed and interfered vector of SNHG16 lentivirus and its negative control were constructed and transfected into ESCC cells to establish the stable cell model of SNHG16 overexpression and interference.The level of SNHG16 in stable cell lines was detected by q RT-PCR,and its efficiency of overexpression and interference was evaluated.2.CCK-8 assay was used to detect the effect on cell proliferation.The colony growth ability of tumor cells was detected by colony formation.Transwell and scratch analysis were used to observe cell migration.3.The established stable cell line was used for tumorigenesis in nude mice in vivo to observe its effect on the tumorigenicity of ESCC cells.Results:1.Lentiviral vectors that overexpressed and interfered with SNHG16 were successfully constructed and transfected into different ESCC cell lines to obtain stable cell lines,in which Eca109 and KYSE140 were used as stable overexpressed SNHG16 cell lines,and KYSE30 and KYSE410 were used as stable interfered SNHG16 cell lines.q RT-PCR analysis showed that compared to controls(LV-NC),the expression of SNHG16 in Eca109 cells(LV-SNHG16)was upregulated by 9.13 times(P<0.05),while by 3.77 times(P<0.05)in KYSE140 cells.However,in the group of KD-SNHG16,the level of SNH16 in KYSE30 cells was 0.22 times lower than that of controls(KD-NC),while 0.24 times in KYSE410 cells(P<0.05).2.CCK-8 showed that OD values of Eca109 and KYSE140 cells overexpressing SNHG16 were higher than those of the control groups(P<0.05).The significant differences of Eca109 cells and KYSE140 cells were shown at 72 h and 48 h,respectively.Colony formation assay showed that the number of cells overexpressing SNHG16 was much more than the controls(P<0.05).Transwell assay showed that the number of transmembrane cells in overexpressing SNHG16 groups was more than the control groups(P<0.05).Scratch test showed that the cell spacing of ESCC cells overexpressing SNHG16 was more narrow than that of the control group(P<0.05).On the contrary,compared with the negative control,the OD value,colony formation number and cell penetration number of ESCC cells in stably interfering SNHG16 group were decreased(P<0.05),while the cell spacing was broadened(P<0.05).3.Subcutaneous tumor-forming assay showed that the tumor weight and volume were significantly higher in the group of overexpressing SNHG16 compared with negative control groups(P<0.05).At the same time,the subcutaneous tumor weight and volume in group of stable SNHG16 interfered were significantly lower than those negative controls(P<0.05).HE and IHC assays showed that the expression of Ki-67 and CD34 in the group of overexpressed SNHG16 was significantly enhanced than that of the control(P<0.05).On the contrary,the expression of Ki67 and CD34 in SNHG16 interfered group was significantly reduced than that of the control group(P<0.05).Conclusions:SNHG16 significantly promotes the proliferation and migration of ESCC in vivo and in vitro,which may play an oncogene role in the occurrence and development of ESCC.Part Ⅲ.SNHG16 combined with EIF4A3 regulate Rhou mRNA stabilityAims:To preliminarily explore whether SNHG16 can bind to RNA binding protein(RBP),intending to further investigate its oncogenic mechanism in ESCC cells.Methods:1.Subcellular fractionation location and FISH assays were analyzed to explore the intracellular localization of SNHG16 in ESCC cell lines.2.The full-length of SNHG16 sense and antisense were constructed,and transcribed in vitro.The products of RNA-protein pull down were stained with silver,and the obtained differential silver bands were detected by protein spectrum(LC-MS/MS).To preliminary analyzed and identified the proteins that could specifically interact with SNHG16.3.Based on the online database analysis and LC-MS/MS,EIF4A3 was identified as the target that interacts with SNHG16.Technique of RIP,RNA-protein pull down,western blot,and q RT-PCR are used to verify the correlation between SNHG16 and EIF4A3.4.Transcriptome sequencing was performed on ESCC cells of sh-SNHG16 and si-EIF4A3 and corresponding controls to analyze the overlapping mRNAs.And verify the co-acting factor of SNHG16-EIF4A3 in the interfering cell lines by q RT-PCR.5.1 μg/μl actinomycin D was added to the cell culture medium of different groups(si-NC,si-EIF4A3,sh-NC,sh-SNHG16,LV-NC,LV-SNHG16,LV-SNHG16+si-EIF4A3).After actinomycin D co-culture for various time points(0h,3h,6h,9h),the remaining of Rhou mRNA was detected by using q RT-PCR.Results:1.Subcellular fractionation assay showed that SNHG16 was mostly located in the cytoplasm against nucleus(P<0.05);FISH staining also showed that SNHG16 was distributed in both cytoplasm and nucleus of ESCC cells,but the fluorescence expression in cytoplasm was significantly higher than that in nucleus(P<0.05).2.Pull-down and silver stain showed specific bands appeared in the scope of 40-55 k Da,35 k Da,and 15 k Da.We performed LC-MS at 40-55 k Da to look for specific RBPs interacting with SNHG16,and discovered 341 differential RBPs binding to SNHG16 sense and 164 RBPs binding to SNHG16 antisense,including 110 overlapping RBPs.3.The specific binding of SNHG16 to EIF4A3 was confirmed by RIP and pull down assays.Western blot and q RT-PCR showed that there were no significant changes in mRNA and protein levels of EIF4A3 after overexpression or interference of SNHG16(P>0.05).Besides,the level of SNHG16 in ESCC cells did not change after EIF4A3 knock down(P>0.05).4.Transcriptome high-throughput sequencing showed that 20,456 genes were differentially expressed in the sh-SNHG16 group compared with the sh-NC group.Meanwhile,there are 22,602 differentially expressed genes in si-EIF4A3 group compared with si-NC.204 differentially expressed genes were found in the overlapped region,and the common downregulated genes include Rhou,FOXO6,WNT4,ST6GALNAC1,AGR2,P4 HTM,NELL2,and ALPP.Among these 8 genes,only Rhou was down-regulated in both cell lines.5.Interference with EIF4A3 shortened the half-life of Rhou mRNA in ESCC cells and reduced its stability(P<0.05).After SNHG16 knockdown,the half-life of Rhou mRNA in ESCC cells was also shortened,which implied decreased mRNA stability(P<0.05).However,The Rhou mRNA stability was not improved after SNHG16 overexpression(P>0.05).Transfected with si-EIF4A3 in stably overexpressing SNHG16 cells,Rhou mRNA stability of cells was decreased(P<0.05).Co-interfered of SNHG16 and EIF4A3,the Rhou mRNA stability of ESCC cells was further reduced(P<0.05).6.EIF4A3 could co-precipitate with Rhou mRNA,and SNHG16 could enhance the co-precipitation of EIF4A3 protein and Rhou mRNA.Interference with SNHG16 and EIF4A3 could reduce Rhou protein in ESCC cells.Conclusions:SNHG16 can bind to EIF4A3,form a complex to regulate the stability of downstream factor Rhou and its mRNA.Part Ⅳ.SNHG16-EIF4A3-Rhou axis in ESCC cellsAims:To investigate the axis of SNHG16-EIF4A3-Rhou in ESCC cells.Methods:1.Si-EIF4A3 was constructed and transient transfected into ESCC cells to observe the effects of si-EIF4A3 on the the ability of cell proliferation,colony formation and migration of ESCC cells by diverse tests.2.Si-Rhou was constructed and transient transfected into stable ESCC cells overexpressing SNHG16 to observe whether si-Rhou affected on the the ability of cell proliferation,colony formation and migration of ESCC cells stable overexpressing SNHG16.Results:1.CCK-8 results showed that the OD values of cells in si-EIF4A3 group were significantly lower than those in the negative control group(P<0.05).Colony formation showed that the number of clones in si-EIF4A3 group significantly smaller than that of NC group(P<0.05).Transwell assay showed that the number of transmembrane cells in si-EIF4A3 weaker than the control group(P<0.05).Scratch assay showed that the spacing of ESCC cells in si-EIF4A3 group was wider than that of the control group(P<0.05).2.CCK-8 showed that the OD value of ESCC cells in LV-SNHG16+si-Rhou group was lower than that of the control group(P<0.05).Colony formation showed that the number of colony formation in vitro of LV-SNHG16 cells transfected with si-Rhou significantly decreased compared with NC group(P<0.05).Transwell assay showed that the number of cell transmembrane of LV-SNHG16 cells transfected with si-Rhou decreased compared with the controls(P<0.05).Scratch assay showed that the spacing of LV-SNHG16 cells transfected with si-Rhou was wider than that of NC group(P<0.05).Conclusions:Rhou attenuate the carcinogenic effect of SNHG16 on ESCC cells.