The Regulatory Mechanisms Of LncRNA-ROR In Hepatocellular Carcinoma Radioresistance

Background and objectiveHepatocellular carcinoma(HCC),with high incidence and mortality rates,is one of the most common malignancies.New HCC cases in China account for about 55%of the total new cases worldwide every year.Moreover,it is the second leading cause of cancer death in our country.Nowadays,radiotherapy is commonly useful for patients with HCC.However,due to the development of resistance,radiation therapy plays a limited role in the treatment of HCC.DNA is a major target of irradiation.Therefore,DNA repair may play an important role in inducing radioresistance in cancers.The function of DNA repair pathway is still unclear.It is clinically valuable to elucidate the mechanisms involved in HCC radioresistance.Currently,dysregulation of long non-coding RNAs(lncRNAs)have been found to serve as a fundamental aspect of cancer biology.LncRNAs mediate the expression of target gene via epigenetic transcriptional regulation or mRNA processing.Recent studies demonstrated that IncRNAs can function as competing endogenous RNAs(ceRNAs)by competitively sponging microRNAs(miRNAs)and binding with the mRNA response element(MRE)present in the miRNAs,thereby regulating gene expression.In our previous research,we proved that linc-ROR can promote the migration and invasion of HCC cells via inducing epithelial-mesenchymal transition,indicating that linc-ROR is a potential therapeutic target for treatment of HCC.Nonetheless,the function of linc-ROR related to radioresistance in HCC is not revealed.This study aims to uncover biological roles of linc-ROR in pathway related to HCC radioresistance.Materials,methods and resultsPart Ⅰ The association of linc-ROR in HCC radioresistanceMaterials,methods1.The HCC cells with radioresistant capacities were established,and then named HepG2-R and SMMC-7721-R.Next,they were validated via colony-forming assays.2.Quantitative real-time PCR analysis(qRT-PCR)was used to measure the expression of linc-ROR in HCC parental cell lines and HCC radioresistant cell lines.Results1.With the increasing doses of IR,the survival rates of the radioresistant HCC cell lines were obviously higher than those of their parental cells,indicating that HepG2-R and SMMC-7721-R acquired increased resistance to radiation.2.The expression of linc-ROR was significantly upregulated in HepG2-R and SMMC-7721-R when compared to their parental cells(p<0.001),indicating that linc-ROR is related to HCC radioresistance.Part Ⅱ The role of linc-ROR in promoting HCC radioresistanceMaterials,methods1.Lentiviruses used to regulate the expression of linc-ROR were established.LV-linc-ROR was used to infect HepG2 and SMMC-7721,and LV-RORsh1 and LV-RORsh2 were used to infect HepG2-R.Then,qRT-PCR was performed to analysis the infection efficiency.2.After stimulation with 4 Gy IR,HepG2/linc-ROR and SMMC-7721/linc-ROR were validated via colony-forming assays.Subcutaneous xenotransplanted tumors were established by injected HepG2/linc-ROR to observe the radioresistant effect of linc-ROR in vivo.3.After stimulation with 4 Gy IR,HepG2-R/RORsh1 and HepG2-R/RORsh2 were validated via colony-forming assays.4.Western blotting and immunofluorescence assay was used to measure the expression of y-H2AX protein in HepG2、HepG2-R、HepG2/control and HepG2/linc-ROR after stimulation with 4 Gy IR.Results1.The expression of linc-ROR in HepG2/linc-ROR and SMMC-7721/linc-ROR were upregulated(p<0.001).Meanwhile,linc-ROR expression decreased in HepG2-R/RORsh1 and HepG2-R/RORsh2(p<0.001).These results demonstrated that the regulation of linc-ROR via lentiviruses in different HCC cell lines is stably.2.Following linc-ROR overexpression,the colony number of HepG2/linc-ROR and SMMC-7721/linc-ROR was significantly more than those of the control HCC cells after stimulation with 4 Gy IR.In vivo,tumor weight and tumor volume was obviously increased in linc-ROR-upregulating group when compared with IR treatment.3.Inhibition the expression of linc-ROR led to a decreased survival capacity of HCC cell lines with radioresistance following 4 Gy IR.4.After IR treatment,results of western blotting assays and immunofluorescences showed that y-H2AX levels at 24h obviously decrease in HepG2-R and remain high in their parental cell lines compared with its levels at 5h,indicating that DNA repair activity is increased in HCC radioresistant cell lines.Following linc-ROR overexpression,y-H2AX was quickly diminished after IR treatment.These data suggested that linc-ROR promotes the resistance of radiotherapy via mediating DNA repair.Part Ⅲ The analysis of molecular mechanisms of linc-ROR/miR-145/RAD18 pathway in regulating HCC radioresistanceMaterials,methods1.In our previous study,it was found that linc-ROR harbor two targets of miR-145.We performed qRT-PCR to measure the level of miR-145 in HCC parental cell lines and HCC radioresistant cell lines.After the expression of linc-ROR was regulated,using qRT-PCR to analysis the change of miR-145 levels.2.Luciferase reporter vectors were constructed and then co-transfected miR-145 mimics in HCC cells.The relative luciferase activity was used to determine whether miR-145 and linc-ROR were combined,which can verify the function of linc-ROR as a molecular sponge.3.Western blotting assays was performed to measure the expression of RAD 18 protein in HCC parental cell lines and HCC radioresistant cell lines.To verify whether linc-ROR can mediate the expression of RAD18,using western blotting to analysis the change of RAD 18 protein levels after the expression of linc-ROR was regulated in HCC cell lines.Following upregulation of RAD18,colony-forming assays were performed after stimulation with 4 Gy IR.The aim was to uncover the role that RAD 18 played in HCC radioresistance.4.LV-linc-ROR,LV-control,miR-145 mimics and miR-NC mimics was used to transfected or co-transfected HCC cell lines.After transfected,western blotting was uesd to analysis the change of RAD 18 protein levels and colony-forming assays were performed after stimulation with 4 Gy IR.These researches were used to analysis the relationship of linc-ROR,miR-145 and RAD18 in HCC radiation resistance.Results1.The expression level of miR-145 was down-regulated in radioresistant HCC cells(p<0.001).Additionally,when HepG2 and SMMC-7721 cells stably upregulated linc-ROR,miR-145 expression was significantly reduced(p<0.001).Conversely,miR-145 expression level was increased in HepG2-R cells with linc-ROR knockdown(p<0.001),indicating that linc-ROR can negatively regulate the expression of miR-145.2.Luciferase reporter assays showed that miR-145 can combine with linc-ROR(p<0.001).These results provided evidence that linc-ROR served as a molecular sponge of miR-145 in HCC.3.Western blot assays showed that RAD18 protein levels were enhanced in radioresistant HCC cells(p<0.001).Moreover,the expression of RAD 18 protein was positively regulated in HCC cell lines with linc-ROR overexpression but downregulated in HepG2-R stably infected LV-shROR(p<0.001).And colony-formation assay revealed that upregulating the expression of RAD 18 decreased survival rates of HepG2 and SMMC-7721 after exposed to IR(p<0.001).These data showed that linc-ROR can positively regulate the level of RAD18 protein.4.After co-transfecting LV-linc-ROR with miR-145 mimics,expression levels of RAD18 were significantly reduced in HCC cell lines(p<0.001).Conformably,the colony numbers of HCC cell which was co-transfected by LV-linc-ROR and miR-145mimics were obviously reduced(p<0.001).The result suggested that linc-ROR modulated RAD 18 expression by competitively sponging miR-145,thereby inducing radioresistance of HCC cells.Conclusion1.The expression of linc-ROR was upregulated in HCC cell lines with radioresistant capacity.2.Linc-ROR,as an oncogene,could promote HCC radioresistance via mediating DNA repair pathway.3.Linc-ROR could competitively bind to miR-145,and subsequently induces the repression of RAD 18 to enhance HCC cells radioresistance.4.In summary,we analysis the expression of linc-ROR in different HCC cell lines.Then,we uncovered the molecular mechanism for miR-145 induced radiation resistance,whereby linc-ROR modulated RAD 18 through competed binding with miR-145,which is required to DNA repair mechanisms.It is promising that combining the radiation therapy with linc-ROR therapeutics to enhance the efficacy of fighting HCC while avoiding resistance.