New Targets And New Tools For Cancer Treatment

Background:The role of NRF2 in hepatocellular carcinoma(HCC)has been extensively studied.It not only can protect normal cells from the harm of carcinogen,but also shield cancer cells against oxidative stress and chemotherapy.As a transcription factor,NRF2 exerts its function by activating downstream genes.Most of studied NRF2 downstream genes are message RNAs.The reports about Long non coding RNAs(LncRNA)activated by NRF2 are relatively less.In this study,we will delineate the molecular links between NRF2 and LncRNA-LUCAT1 and explore the role of LUC ATI in HCC.Methods:First,we compared the expression patterns of LUCAT1 in two patients with distinct prognosis using RNAseq data of seven clinical sample from these two patients.Then,we examined LUCAT1 expression levels in samples from 147 HCC patient by real-time polymerase chain reaction.Futhermore,in order to study the function of LUCAT1,siRNAs and antisense oligonucleotides were employed to knockdown its expression in SNU475,followed by analysis of several NRF2 downstream genes expression and cell viability.Results:The up-regulation of LUCAT1 in tumor was observed in RNAseq data,and the tumor tissues in the patient who has the worse prognosis have higher LUCAT1 expression.The result of real-time polymerase chain reaction also showed that LUCAT1 was upregulated in tumor.Further analysis indicated that higher LUCAT1 expression was positive correlated with worse overall survival,metastasis and vascular invasion.In the in-vitro knockdown experiment,we found only antisense oligonucleotides had the demonstrable high knockdown efficiency.Knockdown of LUCAT1 in SNU475 cells leads to decrease of MRP 1 expression level and cell growth ability,whereas expressions of NQO1 and HMOX-1 remain unaltered.Conclusion:Our data is suggestive of LUCAT1 as a new potential biomarker for HCC.Targeting LUCAT1 may circumvent the dark side of NRF2.However,further study is needed to fully elucidate the function of LUCAT1 in HCC.Background:Estrogen receptor positive breast cancer accounts for nearly 70%among all breast caner patients.The downstream messenger RNAs of estrogen receptor correlate with the efficacy of endocrine therapy.However,the role of estrogen receptor signaling lncRNAs in endocrine therapy is largly unknown.In this study,we aim to identify estrogen receptor signaling-related lncRNAs and study their role in endocrine therapy.Methods:First,we analyzed an existing microarray dataset set from GEO database,which includes E2 treated MCF-7 cells,ICI 182,780 treated cells and E2 plus ICI 182,780 treated group.The lncRNAs in this dataset were identified by using a re-annotated chip-description-file(CDF)provided by ncFANs v2.Second,we employed limma package in R to calculate the differences of gene expression among three treatment groups and determined the estrogen signaling related lncRNAs.We also predicted the function of lncRNAs by constructing a coding non-coding co-expression network using another dataset about MCF-7 cells from GEO and conducting DAVID analysis.Finally,two microarray datasets of human breast cancer tissues were analyzed to evaluate the clinical significance of estrogen signaling-related lncRNAs.Results:Re-analysis of microarray dataset showed that 33 lncRNAs can be agitated by E2 and reversed by ER antagonist ICI.Those IncRNAs are regarded as estrogen signaling related IncRNAs.One of them is PTPRG-AS1,which has been reported to be overexpressed in breast cancer tissue that is associated with tumor grade and clinic outcome of breast cancer patients.Coding-non-coding co-expression network revealed that 15 of those lncRNAs were associated with mitosis,DNA damage,and DNA repair.In addition,we found 5 of those IncRNAs can suggested the endocrine resistance-free survival,metastasis-free survival and disease-free survival.Conclusion:Estrogen signaling related lncRNAs can be novel biomarkers or therapeutic targets for the treatment of breast cancer.BACKGROUND:Producing antibodies against certain antigen is the one of the most important steps of developing therapeutic antibodies.The classic strategy of making antibody is to immune animal with an antigen of interest,followed by hybridoma fusion and screening.Hybridoma screening is a label intense and time-consuming work.Conventional methods,like FACS and ELSIA,have profound drawbacks such as low through-put or generating false positive or false negative.Therefore,the need of new screening method is urgently needed.METHODS:The new method we developed is based on Celigo Image Cytometer.First,CHO cells expressing mouse CD39 were seeded in the 96 well cell culture plates.The next day,cells were fixed and then incubated cells with hybridoma supernatants,followed by staining with fluorescent secondary antibody and Hoechst nuclear stainingand subsequent scan by a Celigo Image Cytometer.Positive hits identified in Celigo were futher validated by Fluorescence-Activated Cell sorting(FACS).RESULTS:When CHO-CD39 cells were fixed before incubation with hybridoma supernatants,65 positive supernatants were picked out from 672 samples by Celigo.However,the none of these positives is validated by FACS.On the contrary,when living cells were incubated with hybridoma supernatants before fixation,Celigo identified 16 true positive supernatants from 576 samples that are consistent with FACS results.Approximately,it only took 9 minutes to scan 96 samples.Futhermore,we demonstrated that Celigo is able to detect antibody signal even when hybridoma supernatant was 256-fold diluted or the concentration of commercial antibody is only around 5 ng/ml.In addition;we successfully obtained the affinity coefficient Kd values of two antibodies by calculating the Celigo data.The Kd value of in house purified anti-CD39 antibody and commercial anti-CD39 antibody is 8.8nM and 3.0nM,respectively.CONCLUSIONS:This novel hybridoma screening method is accurate,high-sensitive and high-throughput.It may facilitate the development of therapeutic antibodies for cancer.