Tumor Suppressor LKB1 Inhibits Both The MRNA Expression And The Amplification Of HTERC By The Phosphorylation Of Yap In Lung Cancer Cells

Objective: Liver kinase B1(LKB1)is widely recognized as a key tumor suppressor gene,and its mutation inactivation can cause many human diseases.Mutation of LKB1 gene is also an important cause of tumorigenesis.Our previous studies found that the expression of h TERC in bronchial brush cells of patients with lung cancer and precancerous lesions was significantly increased,and positively correlated with the severity of lesions.The expression of LKB1 and h TERT was inversely proportional,and the expression of h TERT protein and gene could be down-regulated by phosphorylation of SP1.However,whether LKB1 and h TERC expression are regulated upstream and downstream in lung cancer cells and which genes regulate the heterotypic amplification of h TERC are unclear.If there is a regulatory relationship between LKB1 and h TERC,how LKB1 affects the expression of h TERC and its heterotypic amplification remains to be elucidated in detail.Yes-associated protein(YAP)is a key effector molecule downstream of Hippo signaling pathway,which regulates signal transduction inside and outside the nucleus through phosphorylation,and acts as a transcriptional co-activator to regulate the activity of target protein transcription factors.Previous studies have shown that LKB1 can regulate its transcriptional activation activity by phosphorylating YAP in gastric cancer to limit tissue overgrowth;Nguyen HB and others believe that LKB1 can phosphorylate YAP to nucleate in cervical cancer,but whether there is a regulatory relationship in lung cancer cells has not been reported.It is well known that telomeres gradually shorten with the increasing proliferation of somatic cells.When telomeres shrink to a certain extent,cells stop dividing and remain stationary.Telomere length and stability determine cell life,and are closely related to cell senescence and canceration.Telomerase is a nuclear protease complex,which has the function of synthesizing telomere sequence and balancing telomere consumption.Human Telomerase RNA(h TERC)is a DNA template for telomere elongation and one of the core components of telomere activity.It is very important for the structure and activity of telomere.Our previous studies found that the h TERC and h TERT genes in bronchial brush cells and pleural effusion cells of lung cancer patients were significantly atypical amplification,while the atypical amplification in most normal somatic cells was extremely rare.Therefore,comprehensively understanding and mastering the heterotypic amplification of h TERC in lung cancer cells and the specific regulatory mechanism can provide a new direction for the development of novel tumor-targeting drugs,and provide new ideas for clinical diagnosis and treatment.Yu P and other studies have shown that YAP is one of the key factors to promote the expression of h TERT in breast cancer.However,whether YAP can regulate the transcriptional expression and atypical amplification of h TERC still needs further elaboration.Based on our previous research results and related reports at home and abroad in recent years,we speculate that LKB1 can induce p YAP to remain in the cytoplasm through phosphorylation of YAP,thus inhibiting YAP entry into the nucleus and exerting transcriptional co-activation,thus losing the ability to up-regulate the expression of h TERC and heterotypic amplification.Methods: First,we detected the expression of LKB1 in lung cancer cell lines and normal bronchial epithelial cells by Western blotting.Western blotting and quantitative real-time polymerase chain reaction(q RT-PCR)were used to detect the expression of LKB1,YAP,p-YAP and h TERC.The expression of YAP and p-YAP in the nuclear plasma of LKB1 protein was detected by nuclear plasma separation experiment and Western blotting experiment.the effect of LKB1 protein on the nuclear plasma distribution of YAP protein was detected by immunofluorescence.The heterotypic amplification of h TERC after YAP interference was detected by fluorescence in situ hybridization.The expression of YAP,p-YAP,YAP and h TERC were detected by Western blotting and q RT-PCR.SPSS22.0 statistical analysis software was used to test and analyze the experimental data,p< 0.05 showed significant difference.Results: 1.LKB1 protein was low expressed in lung cancer cell lines.First,we detected the expression of LKB1 protein in three lung cancer cell lines(NCI-H460,A549,LK2)and normal bronchial epithelial cells(HBE)by Western blotting.We found that the expression of LKB1 protein in lung cancer cell lines was lower than that in normal bronchial epithelial cells.The expression of NCI-H460 was higher in lung cancer cell lines,but lower in A549 and LK2 cell lines.In view of this result,we selected A549 and LK2 cell lines to transfect LKB1 expression plasmid and Si RNA to interfere with the expression of LKB1 in NCI-H460 cell lines.2.LKB1 up-regulated p-YAP and inhibited the expression of h TERC.We transiently transferred pc DNA3-LKB1-His to A549 and LK2 with low expression of LKB1.Compared with the control group,overexpression of LKB1 increased the expression of p-YAP and inhibited the expression of h TERC,while the expression of YAP did not change.3.Silencing LKB1 down-regulated p-YAP and up-regulated h TERC expression.After interfering with endogenous LKB1 in NCI-H460 cells,compared with the control group,p-YAP decreased significantly,h TERC gene increased,and YAP protein increased significantly,but YAP gene expression remained unchanged.4.LKB1 could promote nuclear rejection of YAP.To determine whether the localization of YAP is related to the regulation of LKB1,we used Western Blotting to detect the expression of YAP and p-YAP in nuclear plasma.The subcellular expression of YAP was examined by immunofluorescence.After transfection of LKB1,the expression of p-YAP and YAP in cytoplasm increased significantly.YAP in nucleus decreased significantly,but p-YAP could hardly be detected.After interfering with LKB1,the expression of p-YAP and YAP in cytoplasm decreased significantly.YAP in the nucleus was significantly increased,but p-YAP could hardly be detected.However,the expression of YAP was almost absent after transfection and interference with LKB1,so we speculated that LKB1 phosphorylated YAP could inhibit its nucleation.At the same time,we used immunofluorescence test to verify this hypothesis.Immunofluorescence results showed that the expression of YAP in cytoplasm significantly increased after transfection of LKB1.After interfering with LKB1,we found that YAP was significantly expressed in the nucleus.Conclusion: 1.LKB1 inhibits the expression of hTERC in lung cancer cell lines.2.LKB1 can phosphorylate YAP and retain it in the cytoplasm.3.In lung cancer cell lines,YAP can promote the expression and atypical amplification of h TERC.