| Objective Lung adenocarcinoma(LUAD)is the most common subtype of lung cancer with a low average 5-year survival rate.Although the importance of piwi interacting RNA(piRNA)in cancer has been recognized recently,few studies have explored the function and mechanism of piRNA in the occurrence and development of LUAD.The objective of this study was to discover the function and mechanism of piwi interacting RNA piR-hsa-211106 in regulating LUAD progression in vivo and in vitro.Methods The piRNAs differentially expressed in LUAD tissues and adjacent normal tissues were obtained by high-throughput piRNA sequencing.Quantitative Real-time PCR(qRT-PCR)detects the different expression of piR-hsa-211106 in LUAD tissues and cells against adjacent normal tissues and lung epithelial cells at the RNA level.Fluorescence in-situ hybridization(FISH)was used to detect the cellular localization of piR-hsa-211106.Agomir,antagomir of piR-hsa-211106,as well as over-expression and knockdown of piR-hsa-211106 lentivirus,were designed and synthesized respectively,to study their effect on the malignant phenotype of LUAD cells and the sensitivity of chemotherapy in vivo and vitro.Mass spectrometry was used to analyze the pull-down proteins by piR-hsa-211106.RNA binding protein immunoprecipitation(RIP)and RNA pull-down assay were applied to verify their interaction between piR-hsa-211106 and its targeted protein.Finally,qRT-PCR and western blot(WB)were used to detect the regulatory effect of piR-hsa-211106 to downstream target proteins at the m RNA and protein levels.Results(1)qRT-PCR showed that the level of piR-hsa-211106 in LUAD tissues and cells was significantly reduced than that in adjacent normal tissues and lung epithelial cells,and FISH assay suggested that piR-hsa-211106 was localized in both the nucleus and cytoplasm.(2)Agomir promoted the expression of piR-hsa-211106 significantly,and antagomir has a contrary effect.In vitro analysis have shown that over-expression of piR-hsa-211106 can inhibit the proliferation and migration of LUAD cells and promote apoptosis,while knockdown piR-hsa-211106 exhibited the opposite effect.(3)The stable transfected A549 cell constructed by lentivirus can stably overexpress or knock down piR-hsa-211106.The tumor-bearing experiment in nude mice identified that the tumor volume of stably over-expressing piR-hsa-211106 was significantly smaller than that of the control group,while knockdown piR-hsa-211106 was significantly larger than that of the control group.(4)RIP and RNA pull down assays proved that piR-hsa-211106 can interact with pyruvate carboxylase(PC)protein.And over-expression of piR-hsa-211106 can inhibit the expression of PC at m RNA and protein levels,while knocking down piR-hsa-211106 can promote the expression of PC(P<0.05).(5)In vitro analysis shown that cisplatin can inhibit the proliferation of A549 cells in a concentration-dependent manner(20,40,80 μmol/L).Overexpression of piR-hsa-211106 can synergize with cisplatin to inhibit the proliferation of A549 cells and promote apoptosis,while knocking down piR-hsa-211106 reversed the function(P<0.05),proving that piR-hsa-211106 can enhance the chemotherapy sensitivity of cisplatin.(6)In vivo experiments further indicated injection of piR-hsa-211106 agomir can inhibit tumor growth in nude mice and the tumor is inhibited most significantly after treatment with agomir and cisplatin at the same time(P<0.05),which suggested that piR-hsa-211106 can work as a therapeutic target for LUAD.Conclusion In LUAD tissues and cells,the expression of piR-hsa-211106 is significantly reduced than that of adjacent normal tissues and cells.Over-expression of piR-hsa-211106 can inhibit the progression of LUAD,indicating that piR-hsa-211106 is a novel tumor suppressor,which exerts its anti-tumor effect by targeting PC.In addition,piR-hsa-211106 may work as a potential target for the treatment of patients with LUAD,and has a synergistic effect with the chemotherapy drug cisplatin. |
PDF Full Text Request