Preliminary Study On Differential Expression Of Three Kinds Of RNA In Eyelid Sebaceous Gland Carcinoma Based On Whole Transcriptome Sequencing

Objective:As a malignant tumor of ophthalmology,the pathogenesis of eyelid sebaceous gland carcinoma（SGC）is not clear,and little is known about its molecular mechanism.Therefore,the whole transcriptome of eyelid SGC and normal meibomian gland tissue adjacent to cancer were explored based on RNA sequencing（RNA-seq）technology and three types of RNA information were obtained,including m RNA,long non-coding RNA（lnc RNA）and circular RNA（circ RNA）.Method:The clinical data of 3 patients with eyelid sebaceous gland carcinoma after eyelid tumor resection in the Second Hospital of Hebei Medical University were collected.Total RNA was extracted from cancer tissue and normal eyelid gland tissue adjacent to carcinoma during the operation.After passing the quality inspection,a c DNA library was established for high-throughput sequencing of the whole transcriptome.The raw data obtained from sequencing were quality controlled and filtered,and then m RNAs,lnc RNAs and circ RNAs differentially expressed in SGC cancer tissues and normal meibomian glands adjacent to cancer were screened by edge R software.The first five m RNAs,lnc RNAs with significant differential expression were selected and verified by q RT-PCR,relative expression levels were calculated by 2^-△△Ct method.Result:1.Comparison between the eyelid SGC tissues and normal eyelid gland tissues adjacent to carcinoma showed that not only m RNAs and lnc RNAs were differently expressed,but also circ RNAs were abnormally expressed（the screening condition was P<0.05 and the fold change（FC）>1.5）:1)m RNA:A total of 13,010 m RNAs were detected,and 971 m RNAs were differentially expressed,100 were upregulated,and 871 were down-regulated in eyelid SGC tissues compared with normal eyelid gland tissues adjacent to carcinoma.According to the order of FC,the ten upregulated m RNAs with the most significant differences are EIF3CL,TNFAIP8L2-SCNM1,CTAGE4,JMJD7-PLA2G4B,KIAA0408,POTEI,MMP12,SLC24A2,GRM5,HOXB2,and the top ten m RNAs down-regulated are SCGB1D1,LACRT,LCN1,SMR3B,SCGB2A1,PROL1,MYH4,KRTAP3-2,PRR4 and CST4.2)lnc RNA:A total of 8701 long non-coding RNAs were detected,and920 differentially expressed lnc RNAs,including 394 up-regulated and 526down-regulated.According to the ranking of FC value,the top ten up-regulated lnc RNAs are lnc-NEMF-1:9,MALAT1:33,MIR205HG:18,EPB41L4A-AS1:11,LINC00938:6,lnc-ZNF708-2:1,XIST:6,lnc-C21orf58-1:5,lnc-POTEG-14:10 and XIST:64,while down-regulated expression of the top ten are lnc-CCNB1IP1-1:5,NEAT1:23,lnc-ZEB2-1:8,lnc-RNASEH1-7:1,lnc-METTL14-3:13,MALAT1:53,EPB41L4A-AS1:4,lnc-PRR4-1:15,XIST:21 and MIR205HG:12.3)circ RNA:A total of 547 circular RNAs were detected.There were 128differentially expressed circ RNAs in the eyelid SGC tissue,of which 58 were up-regulated and 70 were down-regulated.According to the order of FC value,the top 10 expressions of up-regulation are hsa＿circ＿0005332,hsa＿circ＿0098290,hsa＿circ＿0011169,circular RNA PHRF1,hsa＿circ＿0032116,hsa＿circ＿0108056,hsa＿circ＿0003823,hsa＿circ＿0132667,hsa＿circ＿0137886 and hsa＿circ＿0003234,while hsa＿circ＿0001438,hsa＿circ＿0120806,hsa＿circ＿0001921,hsa＿circ＿0002245,hsa＿circ＿0129072,circular RNA KIAA1211L,hsa＿circ＿0003922,hsa＿circ＿0002882,hsa＿circ＿0003253 and hsa＿circ＿0005077 are the top 10 expressions of down-regulated.2.The first five m RNA,lnc RNA with significant differential expression were selected and verified by q RT-PCR and the results consistent with the differences of sequencing expression were as follows:1)m RNA:The expression level of EIF3CL in eyelid sebaceous gland carcinoma was significantly higher than that in the corresponding normal meibomian gland tissue adjacent to the cancer（t=4.508,P<0.05）.The expression level of SCGB2A1 in eyelid sebaceous gland carcinoma was significantly lower than that in the corresponding adjacent normal meibomian gland tissues（t=-15.530,P<0.01）.2)lnc RNA:The expression level of lnc RNA NEAT1 in sebaceous gland carcinoma of eyelid was significantly lower than that in normal meibomian gland tissue adjacent to the cancer（t=-9.478,P<0.05）.Conclusion:The total transcriptional sequencing technology was used to preliminarily investigate the differences in the expression of m RNAs and lnc RNAs in eyelid SGC compared with the corresponding normal eyelid gland tissues adjacent to carcinoma,and circ RNAs abnormally expressed in eyelid SGC were also found.Differentially expressed RNA might be related to the occurrence and development of eyelid SGC tumors,but the specific functional mechanism needs to be further studied by expanding the sample size.