| Objective:Long non-coding RNAs(lncRNAs)are testified regulators in tumorigenesis and tumor progression.Studies have found that long non-coding SPRY4-IT1 is over-expressed in glioma and has a potential function in glioma cell migration and invasiveness.This study tries to detect the expression level in colorectal cancer and the impacts of SPRY4-IT1 on colorectal cancer cell HT-29,to reveal the function and molecular mechanisms of SPRY4-IT1 in colorectal cancer preliminarily.Methods:1.Fifty-eight surgical samples of colorectal cancer were collected from the Department of General Surgery,the Second Affiliated Hospital of SooChow University(from October 2015 to June 2016),the expression levels of SPRY4-IT1 were testified by qRT-PCR,and the clinical pathological parameters of colorectal cancer were analysized.2.Quantitative real-time polymerase chain reaction(QRT-PCR)was performed to testify the relative expression LncRNA SPRY4-IT1 in the colorectal cancer cell lines(HT-29、HCT-116、SW-480、SW-620、Caco-2)and the normal intestinal epithelial cell line FHC.The study in vitro was performed through selecting HT-29 with the relative highest expression of SPRY4-IT1 Lentivirus-mediated recombinant plasmid target SPRY4-IT1 was constructed and transfected into colorectal cancer cell HT-29 to establish the stably transfected cells of over-expression(up-regulation group)and interference(knock-down group),and negative control group and mock group were established.QRT-PCR was used to testify the relative expression of SPRY4-IT1 in every group cell of HT-29.CCK-8 was applied to detect the proliferation of HT-29.Clone formation assay was used to test the clonality of HT-29.Flow cytometry was applied to detect the apoptosis and cell cycle ofHT-29.Western blot was used to testify the expression of Bcl-2,Bax,Caspase3 and Caspase9.3.Clone formation assay was used to examine the radiosensitivity of HT-29 cells after transfection.CCK-8 assay was to detect the proliferation of post-radiotherapy.The apoptosis,cell cycle and apoptosis-related protein,such asBcl-2,Bax,Caspase3,Caspase9 were detected by flow cytometry and Western blot to analyze the machanism of radiosensitivity.Results:1.The expression of Lnc RNA SPRY4-IT1 in tumor cancer was significantly higher than para-carcinoma tissue,and the higher expression of the colorectal cancer cell lines compared to the normal intestinal epithelial cell line fhc.The expression level of LncRNA SPRY4-IT1 had significant difference among tumor size(P=0.044),infiltration depth(P=0.019),TNM tumor classification(P<0.01),tumor metastasis(P<0.01),serum CEA level(P=0.026),but not having relations with gender(P=0.837),age(P=0.446),tumor location(P=0.425),serum CA19-9(P=0.103).2.The relative expression of LncRNA SPRY4-IT1 in colorectal cancer cell lines was significantly higher than the normal intestinal epithelial cell line FHC,and the expression of SPRY4-IT1 is highest in HT-29(P<0.05).In relative to negative control group and mock group,the proliferation and clonality of the up-regulation group were enhanced,and apoptosis was decreased(P<0.05 or P<0.01),and the proliferation and clonality of knock-down group were reduced,and apoptosis was increased(P<0.05orP<0.01).There was no significant difference in the every group cell cycle of HT-29(P>0.05).In relative to negative control group and mock group,the expression of Bcl-2 was increased and bax was decreased in HT-29 up group(P<0.05orP<0.01),the expression of Bcl-2 was decreased and Bax,Caspase3,Caspase9.was increased in HT-29 down group(P<0.01).3.In relative to negative control group and mock group,the survival fraction of the up-regulation group was higher,and knock-down group was lower.The SER(sensitization enhancing ratio)in up-regulation group were 1.235(D0 ratio)or 1.1957(D0 ratio),the SERin knock-down group were 0.6475(D0 ratio)or 0.6686(D0 ratio).Futher research confirmed that G2/M arrest and apoptosis were induced by 8Gy radiotherapy.The expession of Bcl-2was down-regulated and the expession of Bax,Caspase3,Caspase9 were up-regulated after post-radiotherapy HT-29.Conclusions:1.Our results suggested that LncRNA SPRY4-IT1 severed as oncogenic characteristic in colorectal cancer.2.LncRNA SPRY4-IT1 may significantly accelerate proliferation and restrain apoptosis of colorectal cancer cell via promoting the expression of Bcl-2 and depressing the expression of Bax,Caspase3 and Caspase9.3.The combination of knock-down of SPRY4-IT1 and radio-therapy may significantly restrain tumor growth in colorectal cancer.4.It may be developed as a significant biomarker and treatment target in the diagnosis and treatment of colorectal cancer. |
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