| BackgroundLung cancer is the second most prevalent form of cancer and has the highest mortality rate globally,posing a serious threat to human health.Lung adenocarcinoma(LUAD)is the most common pathological subtype which accounting for approximately 40%of lung cancer,and is known to be the most common cause of death among lung cancer patients.Although various forms of clinical treatments are available for LUAD,including surgery,chemotherapy,targeted therapy,immunotherapy and adjuvant therapy with Chinese medicine,the prognosis of LUAD continues to be unsatisfactory,with a 5-year survival rate of approximately 15%.In recent years,the application of targeted therapy has conferred clinical benefits to the subset of LUAD patients with specific gene mutations.Unfortunately,there is still a large proportion of LUAD patients who do not have mutations of the driver genes which are already known.In addition to this,most LUAD patients had experienced resistance to drugs during the course of molecular targeted therapy.Therefore,identifying new molecular targets is of great importance to improve therapeutic efficacy and the prognosis of LUAD patients.Because of their unique role in signal transduction,protein kinases are major drug targets for many anticancer therapies.UHMK1(U2 auxiliary factor(U2AF)homology motif kinase 1)is a serine/threonine kinase and is the only kinase with a U2AF homology motif(UHM)among the over 500 protein kinases identified thus far.The characteristic structure of UHMK1 enables it not only to be involved in the splicing of pre-mRNAs,but also to participate in multiple biological processes by interacting with other molecules.Specifically,UHMK1 has been identified to interact with the microtubule dynamics regulator protein Stathmin,cyclin-dependent kinase inhibitor(CDKI)p27Kip and the marker of proliferation PIMREG(PICALM interacting mitotic regulator)and so on.In the last few years,many researchers have revealed the function of UHMK1 in promoting the initiation and progression of different types of cancer,including ovarian cancer,melanoma,cervical cancer,liver cancer,colorectal carcinoma,pancreatic cancer and gastric cancer.However,no studies have investigated the function and regulatory mechanism of UHMK1 in LUAD until this day.Therefore,it is necessary to explore the functions and its underlying molecular mechanism of UHMK1 in LUAD which may provide a novel insight for improving the therapeutic effect of LUAD patients.The objectives of the present study were:(1)To explore the expression of UHMK1 in LUAD and its relationship with the clinical characteristics and prognosis of LUAD patients.(2)To investigate the biological functions of UHMK1 in LUAD cells.(3)To identify and validate the downstream target genes of UHMK1 as well as the downstream pathways regulated by UHMK1.Methods1.The Gene Expression Omnibus(GEO)and The Cancer Genome Atlas(TCGA)databases were used to analyze the differences in UHMK1 mRNA expression between LUAD and normal lung tissues and explore the relationship between UHMK1 mRNA expression and the prognosis of LUAD patients.Moreover,the expression of UHMK1 mRNA in the epithelial cells from LUAD tissues and normal lung tissues was analyzed using the single cell sequencing data from the GEO database.2.The expression of the UHMK1 protein in LUAD tissues and adjacent normal lung tissues were measured via tissue microarray-based immunohistochemistry(IHC)and Western blot analysis(WB),and the relationship between UHMK1 protein expression and the clinical prognosis of lung adenocarcinoma patients was analyzed according to the histochemistry score(H-score).3.The expression of the UHMK1 mRNA and UHMK1 protein in LUAD cell lines was detected by real-time quantitative reverse transcription PCR(qRT-PCR)and WB assays.The highest UHMK1-expressing cell line H1299 and the lowest UHMK1-expressing cell line A549 were selected to transfect with UHMK1 knockdown and overexpression lentiviruses,respectively.Then,the stable transduced cells were screened with puromycin.4.CCK-8(cell counting kit 8),EdU(5-ethynyl-2’-deoxyuridine)and clone formation assays were used to detect the effect of UHMK1 on the proliferation of LUAD cells.Moreover,the effects of UHMK1 on the cell cycle and apoptosis of LUAD cells were examined by flow cytometry.The influence of UHMK1 in the autophagy of LUAD cells was observed by transmission electron microscopy.The wound healing and Transwell assays were used to detect the effects of UHMK1 in the invasion and migration abilities of LUAD cells.Furthermore,the effect of UHMK1 on the epithelial mesenchymal transition(EMT)of LUAD cells was detected via WB assay.5.The subcutaneous tumor xenograft model and tail vein injection lung metastasis model were established to assess the effects of UHMK1 on the proliferation and metastasis of LUAD cells in vivo.6.Transcriptome sequencing and bioinformatics analysis were used to screened the downstream target gene UAP1(UDP-N-acteylglucosamine pyrophosphorylase 1)and downstream PI3K(phosphatidylinositol 3-kinase)/AKT(protein kinase B)/mTOR(mammalian target of rapamycin)signaling pathway of UHMK1,which was then validated by WB assay.7.The expression of UAP1 protein in LUAD and adjacent normal tissues were detected by IHC assay using tissue microarray,and the relationship between UAP1 protein expression and the clinical prognosis of LUAD patients was analyzed according to the H-score results.CCK-8,Transwell and WB assays were used to detect the effects of UAP1 on the malignant biological behaviors of LUAD cells and the PI3K/AKT/mTOR signaling pathway.8.A series of rescue experiments were conducted to further confirm that UHMK1 promoted the malignant biological behaviors of LUAD cells by regulating the UAP1mediated PI3K/AKT/mTOR signaling pathway.Results1.UHMK1 expression was up-regulated in LUAD tissues and was positively correlated with a poorer prognosis of LUAD patients.The analysis of the data from the GEO and TCGA databases revealed that UHMK1 mRNA expression was significantly higher in LUAD tissues than that in normal lung tissues.Meanwhile,UHMK1 mRNA expression was positively correlated with a poorer prognosis in LUAD patients.In addition,the analysis of single-cell transcriptome sequencing data showed that UHMK1 mRNA was more significantly expressed in the epithelial cells from LUAD tissues compared with the normal lung tissue epithelial cells.The results of WB and tissue microarray IHC assays demonstrated that UHMK1 protein was highly expressed in LUAD tissues.Furthermore,survival analysis showed that the patients of LUAD with higher expression of UHMK1 had a shorter overall survival(OS)time than those with lower expression.2.UHMK1 promoted the proliferation,migration and invasion while inhibited the autophagy and apoptosis of LUAD cells in vitro.The results of CCK-8,EdU and clone formation assays showed that the proliferation ability of H1299 cells was significantly reduced following UHMK1 knockdown,while was significantly enhanced after the overexpression of UHMK1 in A549 cells.Flow cytometric analysis showed that the knockdown of UHMK1 led to a rise in the proportion of apoptotic H1299 cells as well as a significant G1/S phase block.In contrast,the overexpression of UHMK1 led to a decrease in the proportion of apoptotic A549 cells and accelerated the transition from the G1 phase to S phase.In addition,transmission electron microscopy illustrated that UHMK1 knockdown led to a rise in the number of autophagosome and autolysosome in H1299 cells.In contrast,upregulation of UHMK1 was found to significantly decrease the number of autophagosome and autolysosome in A549 cells.The results of wound healing assay and Transwell assay demonstrated that UHMK1 knockdown inhibited the migration and invasion abilities of H1299 cells,while overexpression of UHMK1 significantly enhanced the migration and invasion abilities of A549 cells.Meanwhile,the WB assay showed that UHMK1 knockdown decreased the expression of mesenchymal markers N-cadherin and Vimentin while increased the epithelial marker E-cadherin.The opposite trend was observed following the overexpression of UHMK1 in A549 cells.3.UHMK1 promoted the proliferation and metastasis of LUAD cells in vivo.The result of the subcutaneous xenograft tumor model in nude mice demonstrated that the tumorigenic ability of H1299 cells was reduced following the knockdown of UHMK1.The tumor volume and weight in the sh-UHMK1 groups were significantly smaller than those in the negative control group.Meanwhile,the tumorigenic ability of A549 cells was observed to be enhanced after the overexpression of UHMK1.The tumor volume and weight in the oe-UHMK1 group were noted to be significantly higher than those in the negative control group.The tail vein injection lung metastasis model showed that the metastatic ability of H1299 cells after the knockdown of UHMK1 was weakened,the number of cancer foci metastasized to the lungs was significantly reduced.In contrast,the metastatic ability of A549 cells following the overexpression of UHMK1 was found to be significantly enhanced,the number of cancer foci metastasized to the lungs was significantly increased.4.The expression of UAP1 was affected by UHMK1.Transcriptome sequencing was performed on stable UHMK1 knockdown H1299 cells together with the negative control cells.The analysis of the transcriptome sequencing data showed that the knocking down of UHMK1 induced the alterations of multiple signaling pathways including the PI3K/AKT/mTOR signaling pathway.The results of WB assay showed that expression of PI3K,phosphorylated AKT(p-AKT)and phosphorylated mTOR(p-mTOR)was inhibited by UHMK1 knocking down in H1299 cells,while overexpression of UHMK1 in A549 cells resulted in the up-regulation of these proteins.The results of transcriptome sequencing,bioinformatics analysis and qRT-PCR experiment indicated that the expression of UAP1,P4HA1,STC1 and GMFB were affected by UHMK1.The result of correlation analysis showed that UHMK1 mRNA and UAP1 mRNA had the strongest correlation in expression among the above candidate genes.5.UAP1 protein was upregulated in LUAD tissues and promoted the proliferation,invasion and migration of LUAD cells.The result of IHC on tissue microarray demonstrated that the expression of UAP1 protein was significantly higher in LUAD tissues than that in the adjacent normal lung tissues and the prognosis of LUAD patients with higher UAP1 expression had a worse prognosis than that of patients with a lower UAP1 expression.The results of CCK-8,Transwell and WB assays showed that the knock-down of UAP1 in H1299 cells impaired the proliferation,invasion,migration and EMT abilities and inhibited the activation of PI3K/AKT/mTOR signaling pathway.6.UAP1 was involved in UHMK1-mediated malignant biological behaviors of LUAD cells.The results of a series of rescue experiments showed that UAP1 could reverse the UHMK1-mediated phenotypic changes and the alterations of the PI3K/AKT/mTOR pathway in LUAD cells.Conclusions:1.The expression of UHMK1 is up-regulated in LUAD and correlates with a poorer prognosis of LUAD patients.2.UHMK1 promotes the malignant biological behaviors of LUAD cells both in vitro and in vivo.3.UAP1 is highly expressed in LUAD and the patients with a higher UAP1 expression had a worse prognosis.4.UHMK1 promotes the malignant biological behaviors of LUAD cells by regulating the UAP1-mediated PI3K/AKT/mTOR signaling pathway. |
