Long Non-coding RNA BANCR Regulates Cancer Stem Cell Markers In Papillary Thyroid Cancer Via The RAF/MEK/ERK Signaling Pathway

[Background and Purpose]Long non-coding RNA is a class of nucleotide sequences with molecular weight more than 200 nucleotides,Because lacking of open reading frame,it cannot be used to encode proteins,but it can influence the occurrence and development of many kinds of tumors by interacting with other related functional genes.The BRAF-activated non-coding RNA(BANCR)was first identified in melanoma,through activating the MAPK/ERK and MAPK/JNK signaling pathway to promote the occurrence and proliferation of melanoma.Recent evidence indicates that these events occur due to the presence of a specific population of cancer stem cells(CSCs),which exist within various tumor types these are considered the root cause of tumorigenesis,metastasis and recurrence.Research shows that BANCR in thyroid carcinoma can promote the cell proliferation and inhibiting the apoptosis,and we have demonstrated that the self-renewal frequency of CSCs in thyroid carcinoma is associated with poor prognosis in thyroid cancer patients.Therefore,this study aims to explore the molecular mechanisms of BANCR regulation of CSC expression in PTC,and potential regulation through the MAPK signaling pathway.[Methods]Firstly,the expression level of BANCR,in thyroid carcinoma tissues,adjacent tissues,and six thyroid cancer cell lines was detected by means of qReal-time PCR.The BANCR-up and BANCR-down cell lines were constructed by using lentivirus,and the transfection efficiency in the six cells was verified by qRea-time PCR.We detected that BANCR obviously affects the proliferation by CCK-8.BANCR-up and BANCR-down cells were injected into mice to observe the tumor formation.Through western blot analysis and flow cytometry to reveal the reflect of BANCR in the expression of the cancer stem cell makers LGR5 and EpCAM.By single-clone formation and microsphere formation experiments to detect the effect of BANCR in increasing the number and size of spheres compared with the control cell line.Western blot was used to find changes in the expression levels of c-Raf,MEK1/2,Erkl/2 and p-c-Raf,p-MEK1/2,p-Erkl/2.Then,the expression of the related markers of CSCs about LGR5 and EpCAM and the phosphorylated expressionof c-Raf,Erkl/2 and MEK1/2 in the Raf-MEK-Erk cell signaling pathway was detected under the stimulation of the inhibitor U0126.[Result]We observed that BANCR is expressed at a higher level in human thyroid tumor tissues than in adjacent normal tissues.The expression level of BANCR differed between cultured thyroid cancer cell lines;BANCR expression was lower in BCPAP,CAL-62,and KMH-2 cell lines than that observed in WRO and FTC-133 cell lines.Interestingly,BANCR expression level was highest in the NPA cell line.Western blot analysis and flow cytometry revealed that over-expression of BANCR in the BCPAP cell line resulted in increased expression of the cancer stem cell makers LGR5 and EpCAM.Alternatively,decreased expression of BANCR via RNA interference in NPA cells resulted in the opposite effect.Single-clone formation experiments showed that upregulated expression of BANCR in the BCPAP cell line promoted an increase in the number of clones formed,while down-regulation of BANCR in the NPA cell line decreased clone formation.Similarly,in microsphere formation experiments,overexpression of BANCR resulted in increased number and size of microspheres compared with the control cell line,where down-regulation of BANCR in the NPA cell line resulted in the opposite effect.Western blotting experiments showed that BANCR over-expression in BCPAP upregulated the expression of phosphorylated c-Raf,MEK1/2,and ERK1/2 where BANCR down-regulation in NPA cells decreased phosphorylation of these proteins.Inhibition of c-Raf via U0126 decreased the expression of LGR5 and EpCAM,as well as phosphorylation levels of c-Raf,MEK1/2,and ERK1/2 in both BCPCP and NPA cell lines,compared DMSO controls.In the xenograft mouse model,BANCR overexpression in PTC cells significantly increased tumor growth.[Conclusion]These results suggest that BANCR plays a role in PTC development by regulating the expression of cancer stem cell markers LGR5,EpCAM via the c-Raf/MEK/ERK signaling pathway.