SSRP1 Promotes Lung Cancer Progression By Blocking The WNT Pathway And Is Negatively Regulated By MiRNA-28-5p

Background:Lung cancer is one of the malignant tumors with the highest morbidity and mortality worldwide.And the 5-year survival rate of patients with lung cancer is low.All of these features listed above make lung cancer still a serious global public health problem.The pathogenesis of lung cancer is very complex and has not been fully elucidated.Current research focuses on finding new tumor metastasis-related markers and exploring more effective targeted and immunotherapeutic methods.SSRP1 has been proven to participate in various biological processes such as DNA transcription,replication,repair,and cell cycle regulation,and play an important role in the occurrence and development of various tumors such as hepatocellular carcinoma,breast cancer,and colon cancer.A large amount of evidence shows that SSRP1 may be a potential target for tumor therapy.DNA repair confers resistance to tumor cells against DNA-damaging anticancer therapies,and γH2AX is critical for recognizing and repairing DNA damage.At the same time,the WNT signaling pathway is abnormally activated in a variety of tumors.As a highly conserved small non-coding RNA,mi RNA is a key regulatory molecule in gene expression,which has been reported to play an important role in the proliferation,apoptosis and metastasis of lung cancer.Among them,abnormal expression of mi RNA-28-5p has been confirmed to be associated with tumorigenesis.However,the mechanism of SSRP1 in the development of lung cancer and the relationship between SSRP1 and mi RNA in the development of lung cancer are still unclear.Objective:This study intends to explore the molecular mechanism of lung cancer development,invasion and metastasis,develop new molecular targeted therapy strategies,and provide theoretical and experimental basis for preventing the occurrence of lung cancer and improving the prognosis of patients。Methods:1.The biological information databases(UALCAN and GEPIA)were utilized to analyze the expression of SSRP1 in normal lung tissue and lung cancer tissue,and whether the expression of SSRP1 is related to the survival rate of lung cancer patients;at the same time,immunohistochemical staining was used to verify the expression of SSRP1 in different tumor stages of human lung cancer tissues.2.Western blot was used to measure the expression of SSRP1 in a variety of lung cancer cell lines(A549,NCI-H446,H1299,PC9 and LLC)and human normal lung epithelial cells(BEAS-2B,B2B).3.RNAi technology was applied to down-regulate SSRP1,and CCK8,cell growth counting and colony formation assays were used to detect the effects of SSRP1 on the proliferation of NCI-H446 and H1299 cells;Transwell was used to prove the effects of SSRP1 on the migration and invasion of NCI-H446 and H1299 cells.4.Western blot was performed to analyze the effects of SSRP1 on the changes of DNA damage marker γH2AX in NCI-H446 and H1299 cells;q-RT PCR and Western blot were used to detect the changes of SSRP1 on apoptosis and cycle-related genes and proteins in NCI-H446 and H1299 cells.5.The changes of NCI-H446 and H1299 cell cycle and apoptosis were detected by flow cytometry.6.Lung cancer xenograft tumor model was established by using NCI-H446 cells to prove the effect of SSRP1 silencing on lung cancer xenograft tumors in vivo.7.Luciferase expression plasmids containing wild-type or mutant SSRP1 3UTR(report-SSRP1-wt and report-SSRP1-mut,respectively)was constructed and transfected into NCI-H446 cells to further confirm whether SSRP1 is mi RNA-28-5p target gene.8.mi RNA-28-5p mimics and mi RNA-28-5p inhibitor was constructed to further verify the effect of mi RNA-28-5p on SSRP1;mi RNA-28-5p mimics and mi RNA-28-5p inhibitor were transfected into NCl-H446 and H1299 cells and Western blot was used to verify the effect of mi RNA-28-5p on SSRP1.9.CCK8,cell counting and colony formation assays were performed to prove the effects of mi RNA-28-5p on the growth of lung cancer cells.Results:1.Compared with normal tissues and cells,SSRP1 is highly expressed in lung cancer tissues and cell lines,and the overall survival rate of lung cancer patients with high SSRP1 expression is lower than that of patients with low SSRP1 expression.2.The results of CCK8 showed that compared with the NC untreated group,after SSRP1 si RNA treatment in NCI-H446 and H1299 cells for 24,48 and 72 h,the cell viability decreased significantly in a time-dependent manner.The results of cell counting experiment showed that compared with the NC group,the number of cells in the si SSRP1 group was significantly reduced.Colony formation experiments showed that compared with the NC group,the number of clones formed in the si SSRP1 group was significantly reduced.3.The results of flow cytometry showed that compared with the NC group,the number of NCI-H446 and H1299 cells in the G0/G1 phase in the si SSRP1 group were significantly increased.At the same time,the proportion of apoptotic cells in the si SSRP1 group was significantly higher than that in the NC group.4.The results of migration and invasion experiments showed that,compared with the NC group,down-regulating SSRP1 reduced the number of lung cancer cells that migrated and invaded.5.The results of Western blot showed that compared with the NC group,the expressions of p-GSK3β and β-catenin in the total protein extracts of the si SSRP1 group were significantly decreased,and the expression of p-GSK3β and β-catenin in the nuclear protein were also significantly inhibited.At the same time,the results of Western blot showed that the protein level of γH2AXwas significantly upregulated in the si SSRP1 group compared with the NC group.6.Compared with the negative control group,the volume of tumor xenografts in mice was significantly reduced in the si SSRP1 group,the expression of pro-apoptotic gene Bax was significantly up-regulated,the expression of anti-apoptotic gene Bcl-2 was significantly down-regulated,the expression of cycle-related gene Cyclin D1 was decreased,and the expression of P21 was increased.In addition,the expressions of p-GSK3β and β-catenin in the total protein extracts were significantly decreased in the si SSRP1 group.7.The results of the dual luciferase reporter gene assay showed that compared with the NC mi RNA group,only the luciferase activity of the mi RNA-28-5p mimics and 3’ UTR-WT combined treatment group were significantly inhibited.However,the luciferase activity of 3’ UTR-MUT did not change significantly under the same conditions.At the same time,Western blot results showed that compared with the NC group,the expression of SSRP1 in the mi RNA-28-5p mimics group was significantly down-regulated,while the expression of SSRP1 in the mi RNA-28-5p inhibitor group was significantly up-regulated.8.The results of CCK8 showed that compared with the NC mi RNA group,the cell viability of the mi RNA-28-5p mimics group decreased significantly at 48 and 72 h,while the cell viability of the mi RNA-28-5p inhibitor group was significantly higher than that of the NC mi RNA group.Compared with the mi RNA group,the number of cells and colonies in the mi RNA-28-5p mimics group were significantly decreased,while the number of cells and colonies in the mi RNA-28-5p inhibitor group were significantly higher than those in the NC mi RNA group.Conclusion:Inhibition of SSRP1 can block the WNT signaling pathway.On the one hand,it can cause cell cycle arrest by affecting the expression of cycle-related genes;on the other hand,it can regulate the expression of lung cancer oncogenes and tumor suppressor genes,induce DNA damage,and trigger apoptosis,thereby further intervening the tumorgenesis and development of lung cancer,which is negatively regulated by mi RNA-28-5p.