Whole-transcriptome Sequencing Of Intrahepatic Cholangiocarcinoma And Biological Function Of RAB3B

Backgrounds:Intrahepatic cholangiocarcinoma（ICC）is a highly malignant tumor originating from intrahepatic biliary epithelial cells.In the past decade,the overall incidence rate of ICC increased globally,while the long-term prognosis of patients with ICC remains dismal.Most patients with ICC can not undergo surgeries at the initial diagnosis and merely receive chemotherapy or other drugs.Moreover the overall prognosis of patients with ICC undergoing surgical resection is far from satisfactory.The poor prognosis of patients with ICC is mainly due to ICC`s high malignancy,predisposition to recurrence and metastasis,and resistance to conventional chemotherapeutic drugs.Malignant tumors are gene mutation diseases in essence.A thorough understanding of the molecular mechanism of the tumorigenesis of ICC will assist in finding therapeutic targets and therefore improve the prognosis of patients with ICC.Whole-transcriptome sequencing is an emerging technique,which can analyze gene expressions on the whole genome level,and provides a means to collect comprehensive information of RNAs in tumor cells,including m RNA,mi RNA,lnc RNA and circ RNA.Constructing potential competitive endogenous RNA regulatory network has become a way of gene transcription research.The differentially expressed genes related to the development of ICC can be identified by Whole-transcriptome sequencing,and the key genes can be screened by bioinformatics analysis,which will provide insight in the molecular mechanism of ICC,and assist in finding potential diagnostic markers and therapeutic targets.RAB3A,RAB3B,RAB3C and RAB3D,related genes of RAS oncogene family members（RAB）,are mainly expressed in neural and secretory cells.RAB3 family genes have been demonstrated to play an important role in the tumorigenesis and invasion of a variety of tumors.RAB3B is a low molecular weight GTP binding protein in the RAB family and plays a key role in vesicle trafficking.It is also showed to plays an important role in the malignant behaviors and chemoresistance of prostate cancer and hepatocellular carcinoma.However the biological role of RAB3B in ICC has not been reported in the literature.Objectives:1)To identify differentially expressed genes of ICC tumors in Chinese patients using Whole-transcriptome sequencing,construct a competitive endogenous RNA（ce RNA）network and screen key differential genes.2)To analyze the molecular biological role of RAB3B in ICC and its effect on chemotherapeutic drugs.Methods:Part 1:To identify the differential expression of m RNAs,mi RNAs,lnc RNAs and circ RNAs in ICC tumors and adjacent normal tissues using whole-transcriptome sequencing technique,differentially expressed genes were functionally enriched and and the ce RNA network was developed via bioinformatics.To verify and screen the genes of interest from whole-transcriptome sequencing results in ICC cell lines HCCC9810,Hu CCT1 and RBE using RT-q PCR.Immunohistochemical staining of clinical tumor samples associated with TCGA database analysis to assess the expression of RAB3B and its clinical significance in intrahepatic cholangiocarcinoma.Part 2:The biological functions of RAB3B on RBE were studied through si RNA knockdown of RAB3B expression in RBE with Proliferation Assay,Invasion Assay and Wound Healing.Identify the relative molecular pathway of RAB3B using RNA-seq of RBE after RAB3B knockdown.Part 3:To analyze the effect of RAB3B on the chemosensitivity of RBE cells to chemotherapeutic agents,IC<sub>50</sub>and the proliferate activity of RBE under the action of5-FU was analyzed with si RAB3B knockdown.Results:Part 1:Compared with adjacent tissues,a total of 2895 dif-m RNAs,56 dif-mi RNAs,151 dif-lnc RNAs and 110 dif-circ RNAs were detected in ICC tumor by Whole-transcriptome sequencing.The functional enrichment analysis of dif-RNA by GO-BP and KEGG showed that the differentially expressed genes were mainly involved in splicing and r RNA processing.The constructed ce RNA network has two hub genes:mi R144-3p and mi R135a-5p.Up-expressed RAB3B was verified in ICC cell lines HCCC9810,Hu CCT1 and RBE.RAB3B and its upstream regulative gene mi R383-5p were the genes of interest.The TCGA database analysis suggested that RAB3B was up-expressed in pan-cancers and was associated with cancers`sensitivity to chemotherapeutic drugs.Part 2:After knocking down the RAB3B level in RBE by si RNA,the malignant behaviors of RBE were significantly inhibited:compared to the nonsense transfection control group,the cellular activity of the si RAB3B group decreased significantly at 48 h and 72 h（P=0.002,P<0.001）,while the mi R383-5p mimics had no effect on the activity of RBE cells at 48 h and 72 h（P=0.999,P=0.896）.The proportion of proliferating cells in the si RAB3B group decreased from 34.67%in the nonsense transfection control group to 17.52%（P=0.0065）.The average number of cells penetrating the Transwell chamber decreased from 1956 in the nonsense transfection group to 481 in the si RAB3B group（P=0.0128）.The migration rate of cells in the si RAB3B group decreased from 37.48%（72 h）and 67.20%（96 h）in the nonsense transfection group to 17.81%（P=0.0017）and 49.71%（P=0.0139）respectively.Knocking down RAB3B in RBE cells resulted in 1038 differentially expressed m RNAs.Through functional enrichment and literature’s analysis,it was suggested that the biological function of RAB3B may involve HIF-1,cell senescence,and tight junctions signaling pathways.Part 3:si RAB3B decreased the IC₅₀of 5-FU on RBE cells,but had no effect on the IC₅₀of gemcitabine and cisplatin.si RAB3B decreased the IC₅₀of 5-FU from 5.725μg/m L in the nonsense transfection group to 2.409μg/m L（P<0.001）,and the IC₅₀fitted-line shifted significantly downward.si RAB3B enhanced the inhibition of RBE cell proliferation by 5-FU:the proportion of proliferating cells in the si RAB3B+5FU group decreased from 36.86%,34.67%,18.72%,and 17.52%in the Blank group,NC group,NC+5FU group,and si RAB3B group to 8.17%.Conclusions:With whole-transcriptome sequencing on paired samples of intra-hepatic cholangiocarcinoma,we constructed a ce RNA regulatory network for Chinese patients.RAB3B level was found to be expressed in pan cancers and associated with the prognosis of intrahepatic cholangiocarcinoma;knocking down the expression level of RAB3B significantly inhibited the malignant biological behavior of RBE cells and increased the inhibition rate of 5-FU on the proliferation activity of intrahepatic cholangiocarcinoma cells,suggesting that RAB3B might be a potential therapeutic molecular target for intrahepatic cholangiocarcinoma.