Overexpression of LINC00152 promotes the proliferation and colony formation of U251 and U87 cells.
To observe the biological effects of LINC00152 on glioma cells U251 and U87, first, we need to achive exogenous overexpression of LINC00152 through transfection. LINC00152 over-expression vector carries green fluorescence tag, and under the fluorescence microscope, it is found that, U251 and U87 cells express green fluorescence in both group empty vector group (pcDNA3.1(+)) and LINC00152 expression group (pcDNA3.1(+)-LINC00152) (Fig. 1a and b). It is indicated that, overexpression vector enters U251 and U87 cells. RT-qPCR result shows that, in the group of pcDNA3.1(+)-LINC00152, both U251 and U87 cells have over-expression of LINC00152 (Fig. 1c).
We have detected viability of cells in both groups, that is, pcDNA3.1(+) and pcDNA3.1(+)-LINC00152, at 12 h, 24 h, 48 h and 72 h through MTT assay. The results show that, compared with pcDNA3.1(+), for U251 and U87 cells in the group of pcDNA3.1(+)-LINC00152, their cell viability is significantly improved (Fig. 1f and 1 g).
At the same time, we perform plate clone to observe the colony formation ability of two cells in both groups, that is, pcDNA3.1(+) group and pcDNA3.1(+)-LINC00152 group. The results show that, either for U251 or U87 MG cells, compared with pcDNA3.1(+), the cell colony formation rate is significantly improved in group pcDNA3.1(+)-LINC00152(Fig. 1d and 1e).
Over-expression of LINC00152 promotes the subcutaneously transplanted tumor growth in U251 and U87 cells.
In vitro experiment reveals that, LINC00152 over-expression promotes proliferation and colony formation of U251 and U87 cells. For this reason, we have created models of U251 and U87 subcutaneously transplanted tumor to further observe the biological effect caused by LINC00152 over-expression in vivo. The data shows that, at the end point (21 Day), in the models with U251 and U87 subcutaneously transplanted tumor, compared with group pcDNA3.1(+), the tumor volume and weight are significantly increased in group pcDNA3.1(+)-LINC00152 (Fig. 2).
Over-expression of LINC00152 promotes YAP nuclear import at U251 and U87 cells.
YAP is a kind of transcriptional co-activator, and it is located at the cytoplasm during the phosphorylation, and it enters nuclei after dephosphorylation, and takes part in the transcription of target gene, further promoting the cell proliferation. Our research have shown that, there are obvious expression of YAP in cytoplasm and nuclei at U251 and U87 cells in group pcDNA3.1(+); however, in group pcDNA3.1(+)-LINC00152, it is mainly expressed in the nuclei, while the cytoplasm expression isn’t obvious. It suggests that LINC00152 promotes YAP nuclear import(Fig. 3).
LINC00152 promotes U251 and U87 cell proliferation through Src-LATS1-YAP signal pathway, rather than MST1/2-LATS1-YAP signal pathway.
To describe YAP’s upstream signal regulation mechanism, we have observed classical MST1/2-LATS1-YAP pathway. The results show that, compared with cDNA3.1(+), for U251 and U87 cells in group pcDNA3.1(+)-LINC00152, both LATS1 and YAP phosphorylation levels are down-regulated significantly. However, it is quite interesting that, no similar result is observed for MST1/2. It suggests that, the regulation of YAP activity depends on LATS1, rather than MST1/2. At the same time, we also find that, compared with group pcDNA3.1(+), for U251 and U87 cells in group pcDNA3.1(+)-LINC00152, Src phosphorylation is significantly up-regulated ; however, after pretreatment with Src inhibitor 1, LATS1 and YAP phosphorylation level is significantly reversed in group pcDNA3.1(+)-LINC00152. It suggests that, in this study, Src is the upstream regulation target of LATS1-YAP(Fig. 4a ~ 4d).
We have performed plate clone experiment to further observe the role of Src in the promotion of cell proliferation by LINC00152. The results show that, after pretreatment with Src inhibitor 1, the colony formation of U251 and U87 cells is greatlyinhibited in group pcDNA3.1(+)-LINC00152(Fig. 4e and 4f). It suggests that, LINC00152 might inhibit LATS1 and YAP phosphorylation by promoting Src phosphorylation, which further promotes U251 and U87 cell proliferation.