| With the rapid development of large-scale high-throughput sequencing technology,piles of studies have shown that non-coding RNAs(ncRNAs)played a pivotal role in disease regulation and pathological processes.The most frequently studied ncRNAs are the microRNAs and siRNAs which act on RNA interference(RNAi).Long non-coding RNA(lncRNA)is one of the important members of ncRNAs,and its ratio accounts for nearly 80% of all ncRNAs.LncRNAs and their target genes may participate in various activities,including transcriptional and translational regulation,posttranscriptional epigenetic modification,cell cycle control,cell differentiation and apoptosis and so on.The changes of expression level or even spatial structure change of LncRNAs which are widespread in the genome would cause a series of protein expression alterations and changes in signal pathway status.Transcription of RNA from its DNA template is orchestrated by three different complexes termed RNA polymerases(Pols)I,II,and III.While RNA Pol II handles both the transcription of mRNAs and noncoding RNAs,RNA Pol I and RNA Pol III have so far only been found to transcribe noncoding RNAs.In a previous study,we have developed a new and simple strategy to produce siRNA library with an especial vector which contains two opposing RNA polymerase III(U6 and H1).Based on this finding,a genomic DNA-derived nc RNA expression library was designed and constructed by cloning the sonicated-human genomic DNA fragments into an expression cassette driven by converging dual U6 and H1 RNA polymerase III promoters.We hypothesize that such ncRNA library,if providing sufficient genome coverage,should be suitable and sufficient for a genome-wide functional screening of ncRNAs.To ensure the high quality of the library,we constructed the library system by using the highly efficient Gibson Assembly which overcomes the disadvantage of classical molecular cloning technology in the library construction.The assembly efficiency was rather high,and one assembly reaction yielded on average about 2.3 × 106 colonies.A total of 5 Gibson Assembly reactions were carried out,producing about 1.2 × 107 colonies.Colony PCR and DNA sequencing analysis of randomly picked up 8 positive clones confirmed the diversity and representation of ncRNA library.To validate the function of the newly made ncRNA library,we tried to perform functional screens of transcripts whose expression were affected by ncRNA may contribute to the development of drug resistance to BRAFV600 E inhibitor vemurafenib in sensitive or na?ve human melanoma A375 cells.We found the lethal concentration of Vemurafenib for na?ve A375 cells is at 10 μM.We infected A375 cells with packaged ncRNA retrovirus library and the infected A375 cells were subjected to Vemurafenib selection concentration(10 μM).The vast majority of the cells were killed by the Vemurafenib at the early stage,while some clonal recoveries were observed.The survived cells,were pooled,expanded and designated as A375/HL.We found that A375/HL confered Vemurafenib resistance when compared with na?ve A375 cells.Compared with na?ve A375 cells,theVemurafenib-resistant A375/HL cells became narrow and assumed spindle shape-properties with the mesenchymal phenotype.The WST-1 assay showed that the IC50 value of A375/HL(8.76 μM)was higher than that of naive A375 cells(4.17 μM).The abilities of colony formations were much stronger under the presence of 1 μM Vemurafenib.We also found that,compared to na?ve A375 cells,A375/HL cells were resistant to vemurafenib at high concentration(30 μM)after treatment for 24 hours.There were more drug precipitations in A375/HL cell group and these finding,suggesting that A375/HL may pump vemurafenib out more efficiently than na?ve A375 cells.Using qPCR analysis,we compared the expression levels of EMT-related markers between A375/HL and na?ve A375 cells.The expression of epithelial marker E-cadherin was decreased while some other mesenchymal markers were all up-regulated in A375/HL cells,compared with that in na?ve A375 cells.We tested 10 cancer-associated pathway reporter activities in A375/HL with or without vemurafenib treatment and found that there was no activated signal pathway,while vemurafenib inhibited multiple cancer-associated signaling pathways significantly,including Elk1 / SRF、AP1、Myc/Max and STAT in A375/HL cells.It has been widely reported that BRAF inhibition can lead to the adaptive activation of other signaling pathways.We further studied the phosphorylation levels of EGFR super signaling pathway’s key kinases: Akt,STAT3,MEK and Erk.Confocal immunofluorescence staining showed that the expression levels of p-Akt,p-STAT3,p-MEK and p-Erk in A375/HL cells were higher than that of na?ve A375 cells,suggesting that there was compensatory activation of other signaling pathways,including EGFR signaling pathways.Thus,these results demonstrate that vemurafenib-resistant A375 lines(A375/HL)generated by using our ncRNA library share the characteristics of vemurafenib-resistant phenotypes described in other acquired resistance studies using human melanoma lines and/or patient-derived na?ve and drug-resistance tumor samples,indicating that our genome DNA-derived ncRNA library-based screening is a valid approach to the identification of vemurafenib resistance genes in a genome-wide fashion.At last,we extracted the genomic DNA of vemurafenib-resistant cells A375/HL and add sequencing primers through PCR amplification the inserts in the genome to recover the functional ncRNA sequences.The quality and quantity of PCR-amplified ncRNA sequences library were assessed by using Agilent 2100-High Sensitivity DNA Assay Kit before next-generation sequencing.In conclusion,the human genome DNA-derived ncRNA library we constructed is the first report of its kind.The current protocols and strategies we used for the functional screening of drug-resistance relating gene were available.The results presented here have demonstrated this type of ncRNA library may be an important tool for genome-wide screening of functional genes. |
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