Study Of The Effects On Biological Behavior Of MiR-153 In Non-small Cell Lung Cancer A549 Cell By Targeting SRC And Its Mechanisms

Objective: As the leading cause of cancer-related mortality,lung cancer accounts for 25.3% worldwide.Meanwhile,60% of lung cancer are diagnosed at a distant stage and thus makes lung cancer more challenging.Regardless of constant progress in survival for most cancers,the 5-year relative survival rate of lung cancer is 18.1%.Therefore,in order to improve the early diagnosis of lung cancer and reduce the mortality,studying the underlying molecular mechanism and exploring valid therapeutic targets are of great significance.miRNAs are a group of endogenous non-coding RNAs containing 18-25 nucleotides which favor translational repression or degradation of target mRNAs by binding to 3'UTRs of the gene.Increasing evidence demonstrates that miRNAs participate in multiple biological processes including proliferation,survival,angiogenesis and resistance to chemotherapy.Therefore,miRNA has become an important molecular marker of prognosis,development and progression of cancer.Currently,clinical trials using miRNAs as targets for antitumor therapy are progressing.SRC or c-SRC,a classic proto-oncogene,plays a pleiotropic role in the tumorigenesis and metastasis of various cancers by encoding a non-receptor tyrosine kinase.On the molecular mechanism,SRC involves tumor proliferation,apoptosis,migration,and invasion.However,taken SRC inhibitors as monotherapy for cancer is unsatisfactory.Combining SRC inhibitors with other antineoplastic agents may work synergistically.This study intended to explore the roles of miR-153 in lung cancer proliferation,apoptosis,migration,invasion and perfect the function-network of miR-153 in order to explore whether miR-153 can be a therapeutic target against lung cancer.Methods: 1.TargetScan,miRNA.org,mirDIP,miRcode,and Diana Tools were used to predict possible targets of miR-153.LinkedOmics database was used to verify the correlation between miR-153 and target gene expression.Ualcan database was used to explore the expression of SRC in lung adenocarcinoma and lung squamous cell carcinoma.Kaplan-Meier Plotter database was applied to demonstrate the effect of SRC on overall survival of non-small cell lung cancer.2.A luciferase reporter assay was conducted to further confirm the regulation between miR-153 and SRC 3'UTR by constructing SRC 3'UTR-WT and SRC 3'UTR-MUT and being co-transfected with miR-153 mimic or mimic control in 293 T cell.3.miR-153 overexpression,miR-153 inhibition and target gene silencing A549 cell was constructed by transected miR-153 mimic,inhibitor and target gene siRNA.The transfection effect was evaluated by qRT-PCR,the effects on target gene(SRC)protein expression was evaluated by western blot.4.The colony formation assay was applied to examine the effects of miR-153 and SRC on the colony formation ability of A549 cells.5.The CCK8 assay was conducted to explore the effects of miR-153 and SRC on the proliferative activity of A549 cells.6.The EdU assay was used to illuminate the effects of miR-153 and SRC on DNA synthesis of A549 cells.7.Cell apoptosis was examined by Annexin V-FITC/PI staining.8.The effects of miR-153 and SRC on cell migration were verified by wound healing assay.9.The effects on cell invasion were tested by transwell matrigel invasion assay.10.miR-153 inhibitor and SRC siRNA,siRNA control were co-transfected in A549 cells respectively,the EdU assay was applied to explore whether SRC is the functional target gene of miR-153.11.miR-153 inhibitor and SRC siRNA,siRNA control were co-transfected in A549 cells respectively,the transwell matrigel invasion assay was applied to explore whether SRC is the functional target gene of miR-153.12.The effect of miR-153 mimic and target gene siRNA on downstream protein expression was verified by Western blot.13.GraphPad Prism software was applied for statistical analysis.Statistical comparison between groups were analyzed using the unpaired,two-tailed student's t-test,and P<0.05 was considered statistically significant.Results: 1.SRC was identified as a potential target of miR-153 by using Targetscan,microRNA.org,mirDIP,miRcode and Diana Tools.The binding sequences between miR-153 and 3'UTR of SRC are conserved across species.LinkedOmics database demonstrated that the log2-scale expressions of premature miR-153(miR-153-1,miR-153-2)and SRC are negatively correlated in 63 lung adenocarcinoma patients.Ualcan database illustrated that the expression of SRC in lung adenocarcinoma and lung squamous cell carcinoma was up-regulated.Kaplan-Meier Plotter database showed that overexpression of SRC predicted a poor overall survival in non-small cell lung cancer.2.Co-transfection of SRC 3'UTR luciferase vector with miR-153 mimic to 293 T cells showed that the luciferase activity in co-transfection group was significantly lower than that in control group.3.Overexpression of miR-153 or SRC silencing reduced the expression of SRC protein.4.The colony formation assay illuminated that miR-153 mimic or SRC siRNA significantly suppressed the proliferation of A549 cells,while miR-153 inhibitor significantly promoted the proliferation of A549 cells.5.The CCK8 assay demonstrated that miR-153 mimic or SRC siRNA significantly suppressed the proliferative ability of A549 cells,while miR-153 inhibitor significantly promoted the proliferative ability of A549 cells.6.The EdU assay illustrated that miR-153 mimic or SRC siRNA significantly suppressed the DNA synthesis of A549 cells,while miR-153 inhibitor significantly promoted the DNA synthesis of A549 cells.7.The Annexin V-FITC/PI staining illustrated that miR-153 mimic or SRC siRNA significantly promoted the apoptosis of A549 cells,while miR-153 inhibitor significantly inhibited the apoptosis of A549 cells.8.The wound healing assay demonstrated that miR-153 mimic or SRC siRNA significantly suppressed the migration of A549 cells,while miR-153 inhibitor significantly promoted the migration of A549 cells.9.The transwell matrigel invasion assay showed that miR-153 mimic or SRC siRNA significantly suppressed the invasion of A549 cells,while miR-153 inhibitor significantly promoted the invation of A549 cells.10.When compared with the group of miR-153 inhibitor and siRNA control,the DNA synthesis of A549 cells in the group of miR-153 inhibitor and SRC siRNA was significantly decreased as demonstrated by the EdU assay.11.When compared with the group of miR-153 inhibitor and siRNA control,the invasive ability of A549 cells in the group of miR-153 inhibitor and SRC siRNA was significantly decreased as demonstrated by the transwell matrigel invasion assay.12.Western blot exhibited that transfection with miR-153 mimic or SRC siRNA significantly decreased the expression of MMP2 compared with corresponding controls.Conclusion: 1.miR-153 inhibits the expression of SRC by targeting the 3'UTR,and the expression of miR-153 in lung adenocarcinoma is negatively correlated with SRC.And the expression of SRC in lung adenocarcinoma and lung squamous cell carcinoma was up-regulated,overexpression of SRC predicted a poor overall survival in non-small cell lung cancer.2.miR-153 inhibits the expression of SRC protein in A549 cells.3.miR-153 functions as a tumor suppressor in A549 cell by suppressing the proliferation,migration,invasion,and promoting the apoptosis.4.Silencing SRC inhibits the proliferation,migration,invasion,and promotes the apoptosis of A549 cells.5.SRC is a functional target of miR-153.6.miR-153 inhibits the SRC/MMP2 signaling pathway.