CircSPAG16 Suppresses Cadmium-induced Transformation Of Human Bronchial Epithelial Cells By Decoying PIP5K1?

Objective:Cadmium is a chemical pollutant that exists widely in the environment and one of the carcinogens that induce lung cancer.The direct genotoxicity of cadmium is weak,so the epigenetic mechanism has become one of the main mechanisms on the carcinogenic mechanism of cadmium.Circular RNAs(circ RNAs)are a kind of epigenetic molecules that have been found to play roles in physiological and pathological events recently.In recent years,the regulatory roles of circ RNAs in cancers have been gradually investigated.However,in the study of lung cancer caused by cadmium,the role and mechanism of circ RNA are still unclear.Circ RNAs can participate in various of physiological and pathological processes through a variety of mechanisms,such as sponging mi RNA,directly binding with proteins and translating polypeptides or proteins.The mechanism that circ RNAs work as mi RNA sponges has been most frequently investigated.Recent researches discovered that more and more circ RNAs could play a role by directly interacting with proteins.It has been reported that circ RNA participates in the regulation of chemical carcinogenesis through the ce RNA(competing endogenous RNA)mechanism,but it remained unknown whether circ RNA could function through interacting with proteins in chemical carcinogenesis.In vitro cell transformation model is a research model that uses cell culture to simulate the tumorigenicity of carcinogens in vivo and is often used in the research of chemical carcinogenesis.Therefore,with cadmium-induced transformation of BEAS-2B cells as the model,this study aimed to study the role of circ RNA in cadmium-induced human bronchial epithelial cells(BEAS-2B)transformation and the mechanism that circ RNA regulated the malignant phenotypes of cadmium-transformed cells by directly interacting with proteins.Methods:The BEAS-2B cells were continuously treated with 2.0?M cadmium chloride for 20 weeks,and anchorage-independent growth assay was used to detect the anchorage-independent growth ability of the cells in the treatment group and the control group.After cell transformation was done,RNAs were extracted from the control-BEAS-2B(C-BEAS-2B)cells and the transformed BEAS-2B(T-BEAS-2B)cells.Nanodrop spectrophotometer,Agilent 2100 analyzer and 1%agarose gel electrophoresis were used to detect quality of RNAs.Illumina Hi Seq Xten platform was used for high-throughput sequencing.The q RT-PCR(quantitative real-time polymerase chain reaction)was done to verify the expression of the top 5 up-regulated and down-regulated circ RNAs.RNase R and oligo(d T)primers were used to verify the circular characteristics of circ SPAG16.Nuclear and cytoplasmic isolation experiment was used to confirm the subcellular location of circ SPAG16 in BEAS-2B cells.The stability of circ SPAG16 in BEAS-2B cells was detected with actinomycin D treatment.The q RT-PCR detected levels of circ SPAG16's linear isoform in C-BEAS-2B and T-BEAS-2B,the expression changes of circ SPAG16 during the process of cadmium-induced cell transformation,and the comparison of circ SPAG16 in lung cancer cell lines with normal BEAS-2B cells.Using lentivirus vector with full sequence of circ SPAG16 or si RNA specific to circ SPAG16,circ SPAG16 was overexpressed or knock down in T-BEAS-2B cells,then the cancer hallmarks of the transformed cells including cell proliferation,migration,invasion and anchorage-independent growth ability were detected by plate colonies formation assay,cell scratch assay,transwell invasion assay and anchorage-independent growth assay,respectively.RNA pulldown assay,protein qualitative mass spectrometry and RNA immunoprecipitation experiments were used to confirmed that protein PIP5K1?(phosphatidylinositol 4-phosphate5-kinases?)could bind to circ SPAG16.ISA-2011B,a specific inhibitor of PIP5K1?,was used to study the role of PIP5K1?in T-BEAS-2B cells.Circ SPAG16overexpression in T-BEAS-2B cells were done to study the role of circ SPAG16 on PIP5K1?and its downstream PIP₂[phosphatidylinositol(4,5)bisphosphate]and p Akt(phosphorylated Akt).By co-expressing circ SPAG16 and PIP5K1?to reverse PIP5K1?in T-BEAS-2B cells,whether circ SPAG16 functioned through regulating PIP5K1?was checked.Plate colonies formation assay,cell scratch assay,transwell invasion assay and anchorage-independent growth assay were used to detect cell proliferation,migration,invasion and anchorage-independent growth ability.Immunofluorescent staining was used to detect the expression level of PIP₂ and Western blot were used to detect p Akt.Result:results were divided into three parts.Part one:1.High-throughput sequencing detected 9857 circ RNAs,of which 222were significantly up-regulated(P<0.05,fold-change>1.5),and 256 were significantly down-regulated(P<0.05,fold-change>1.5).2.For the q RT-PCR experiments of the top 5 up-regulated and down-regulated circ RNAs,two circ RNAs were excluded for the poor specificity of their primers.Among the remaining 8circ RNAs,half of them were consistent with the sequencing results with statistical significance(P<0.05).3.Circ SPAG16(circ Base ID:hsa?circ?0058040)was the most down-regulated circ RNA in T-BEAS-2B cells.The expression level of circ SPAG16 in T-BEAS-2B cells was approximately one-fifth of that in C-BEAS-2B cells(P<0.01).Part two:1.Circ SPAG16 was more resistant to RNase R compared to SPAG16m RNA(P<0.01).Circ SPAG16 had no poly(A)tail,and was mainly located in the cytoplasm.Compared with linear RNA,circ SPAG16 had higher stability(P<0.05).2.There was no significant change in the level of SPAG16 m RNA between C-BEAS-2B and T-BEAS-2B cells.3.During cadmium-induced transformation,there is a significant descending trend of circ SPAG16 expression at 8,12,16 and 20 weeks(P<0.05 or P<0.01).4.Compared with normal BEAS-2B cells,the expression of circ SPAG16 was significantly down-regulated in lung cancer cell lines A549,H1299 and H460(P<0.05or P<0.01).5.Overexpression of circ SPAG16 in T-BEAS-2B cells significantly inhibited the cancer hallmarks of the cells(P<0.01).On the contrary,knocking down circ SPAG16 significantly enhanced the cancer hallmarks of the cells(P<0.05 or P<0.01).6.Circ SPAG16 inhibited cadmium-induced cell transformation of BEAS-2B cells(P<0.05 or P<0.01).Part three:1.Circ SPAG16 directly interacted with the phospholipid kinase PIP5K1?.2.PIP5K1?promoted the cancer hallmarks of T-BEAS-2B cells through inactivating Akt(P<0.05).3.Overexpression of circ SPAG16 in T-BEAS-2B cells significantly reduced the levels of PIP2 and p Akt(P<0.05).4.Co-expression of circ SPAG16 and PIP5K1?significantly reversed the inhibition of circ SPAG16 on the levels of PIP2 and p Akt(P<0.01),as well as the cancer hallmarks of T-BEAS-2B cells(P<0.01).Conclusions:Circ SPAG16 was the most down-regulated circ RNA in T-BEAS-2B cells relative to C-BEAS-2B cells.Circ SPAG16 inhibited cadmium-induced cell transformation of BEAS-2B cells.Circ SPAG16 inhibited cadmium-induced cell transformation of BEAS-2B cells through directly interacting with PIP5K1?to inactivate Akt.