Microarray Profiling And Functional Screen Analysis Reveal Long Non-coding RNA-SNHG7 As A Target Of MiR-34a To Increase Level Of GALNT7 And Regulating PI3K/Akt/mTOR Pathway In CRC Progression

Objective: The differential expression of lncRNA and mRNA expression profiles in primary colorectal cancer SW480 cell line and metastatic colorectal cancer SW620 cell line was verified by microarray.CNC analysis confirmed the relationship between lncRNA-SNHG7 and m RNA co-expression profiles and elucidated the functional mechanism of lncRNA-SNHG7 in the progression of colorectal cancer,providing a new direction for the early diagnosis and treatment of colorectal cancer.Methods:(1)The expression of lncRNAs in colorectal cancer cell lines(SW480,SW620)was verified by microarray.The expression of differentially expressed lncRNAs in colorectal cancer tissues and cells was detected by qRT-PCR.Screening for lncRNA-SNHG7 associated with metastasis of colorectal cancer The expression of SNHG7 in clinical colorectal cancer samples was detected by qRT-PCR.The relationship between the expression of SNHG7 and clinicopathological parameters and the prognostic significance were analyzed by SPSS17.0 software and Graphpad Prism 5 software;(2)To construct a cell line stably overexpressing LncRNA-SNHG7(SW480-SNHG7)and a control cell line(SW480-vector),siRNA was used to interfere SNHG7 expression and control siSCR.Cell proliferation and proliferation were detected by CCK8,Edu and plate clone assays.Cell cycle and apoptosis were detected by FACS.Wound healing assay,transwell migration and invasion assay were used to detect cell migration and invasion ability.Cell angiogenic ability was measured by angiogenesis assay.Tumorigenicity of colorectal cancer cells in nude mice;(3)In order to discuss the specific molecular mechanism of the effect of LncRNA-SNHG7 on the cell phenotype,we used the biological software Starbase v2.0,luciferase reporter gene,RNA co-precipitation and Western blot,based on the theory of ceRNA regulation,to detected the competitive relationship between SNGH7 and miRNAs.The co-expression of lncRNA-SNHG7 and mRNAs was found by CNC analysis;(4)The effect of overexpressed LncRNA-SNHG7 on PI3 K / Akt / mTOR pathway in colorectal cancer cell lines SW480 and SW620 was detected by Western blot.Results:(1)Microarray expression profiles showed that LncRNA-SNHG7 was highly expressed in the colorectal cancer metastasis cell line SW620 compared to the colorectal cancer in situ cell line SW480,and the clinical sample demonstrated that SNHG7 expression in colorectal cancer tissues was higher than that in the paracancerous tissues.The correlation between LncRNA-SNHG7 expression and clinical phenotype in 39 patients with colorectal cancer was analyzed.The expression level of LncRNA-SNHG7 was significantly correlated with tumor size,lymph node metastasis,distant metastasis and tumor staging.The survival curves of patients with colorectal cancer with LncRNA-SNHG7 overexpression were significantly lower than those without LncRNA-SNHG7.Log-Rank test was used to compare the survival curves between the two groups,which indicated that the overall survival and disease-free survival Lower than the low expression group,the difference was statistically significant;(2)The proliferation of LncRNA-SNHG7 overexpression cells was significantly increased in vitro compared with the control group by the cell proliferation assay of CCK8,Edu and plate clone.FACS showed that LncRNA-SNHG7 overexpression induced cell cycle and inhibited cell apoptosis;Wound healingassay,migration experiments and invasion experiments showed that the migration and invasion ability of LncRNA-SNHG7 overexpression cells was significantly increased;angiogenesis experiments showed that the rate of neovascularization was significantly increased after LncRNA-SNHG7 overexpression;Lnc RNA-SNHG7 expression enhances colorectal cancer cells in vivo tumorigenicity;(3)Starbase v2.0 prediction software predicts that LncRNA-SNHG7 has a targeted relationship with miR-34 a,and this result is verified by dual luciferase reporter assay,RNA co-precipitation assay and Western blot.CNC analysis showed that there was a positive correlation between LncRNA-SNHG7 and GALNT7 expression.Western blot was used to detect SNHG7-overexpressing cell lines.Compared with the control group,the expression of GALNT7 was significantly increased,and the trend was consistent with that of SNHG7.By analyzing the clinical samples RT-PCR results,SNHG7 and GALNT7 showed a significant positive correlation.It is indicated that LncRNA-SNHG7 may be involved in the development of colorectal cancer by competitively binding miR-34 a to promote the expression of proto-oncogene GALNT7;(4)Specific up-regulation of SNHG7 expression can increase the expression of key proteins in PI3 K / Akt / mTOR pathway and promote the activation of the pathway.Conclusion:(1)LncRNA-SNHG7 is highly expressed in colorectal cancer tissues and highly metastatic cell lines,and has significant correlation with tumor size,lymph node metastasis,distant metastasis and tumor staging.LncRNA-SNHG7 is a risk factor for prognosis of colorectal cancer;(2)LncRNA-SNHG7 plays an important role in the proliferation,migration,invasion and angiogenesis of colorectal cancer;(3)LncRNA-SNHG7 may play a regulatory role on the oncogene GALNT7 by competitively binding to miR-34a;(4)LncRNA-SNHG7 affects the activation of PI3 K / Akt / mTOR pathway protein by competitively binding miR-34 a and regulating GALNT7,thereby affecting the development of colorectal cancer.