The Role And Mechanism Of Long Non-coding RNA FAL1 In The Progression Of Colorectal Cancer

Objective:In our study,we plan to investigate the effects and related mechanism of long non-coding RNA FAL1 on the proliferation,EMT,invasion and migration of colorectal cancer cells,and It provides a new direction for the prognosis assessment and targeted therapy of colorectal cancer.Methods:1.We selected 50 groups of clinically resected colorectal cancer specimens and corresponding adjacent tissues.Real-time quantitative PCR(RT-q PCR)was used to analyze the expression of lnc RNA FAL1 in colorectal cancer and adjacent tissues,as well as HCo Epi C and five colorectal cancer cell lines.50 pairs of specimens were divided into high expression group and low expression group as the level of lnc RNA FAL1.According to clinical information,the correlation between lnc RNA FAL1 and clinical data was analyzed,and Kaplan-Meier was used for survival curve analysis.2.We transfected HCT116 and HT29 cells by artificial plasmid vector that overexpressed or silenced lnc RNA FAL1.Then screened the stable overexpressed and silenced cell lines and confirmed the transfection efficiency.Next we observed the impact of lnc RNA FAL1 on cell proliferation,migration and invasion of HCT116 and HT29 cells via MTT assay,colony formation assay and transwell assay.The effects of lnc RNA FAL1 on epithelialization(EMT)-related factors E-cadherin and Vimentin were determined by RT-q PCR and western blot.3.We predicted the targeted mi RNA of lnc RNA FAL1 by searching Reg RNA website.Then dual luciferase reporter assay and RT-q PCR were play to verify the direct targeting relationship.RT-q PCR was used to detect the expression of mi R-637 in 50 pairs of colorectal specimens.The Pearson correlation test was used to analyze correlation between mi R-637 and lnc RNA FAL1 in colorectal cancer tissues.Furthermore,HCT116 and HT29 cells were transfected by mi R-637 mimics and ASO-mi R-637.And then the proliferation ability,EMT related factors,invasion and migration ability were measured.4.We further predicted the potential targeted gene of mi R-637 by searching Target Scan 7.1 website.Then the same assays were used to validate the direct targeting relationship between mi R-637 and NUPR1.We also found the correlation between mi R-637 and NUPR1 in colorectal cancer tissues.After transfected by NUPR1-overexpressed or NUPR1-silenced plasmid,the proliferation ability,EMT related factors,invasion and migration ability were also measured.5.It was found by literature reports that there is a correlation between NUPR1 and HIF-1α,which is also associated with LASP1.RT-q PCR and western blot were used to detect the expression levels of HIF-1α and LASP1 in cells overexpressed or silenced of NUPR1.The effects of overexpressing NUPR1 meanwhile silencing HIF-1α on the malignant phenotypes of HCT116 and HT29 cells were analyzed.6.Simultaneously overexpressed of Lnc RNA FAL1 and mi R-637 and observed the changes in proliferation,invasion,migration abilities of transfected HCT116 and HT29 cells..RT-q PCR and western blot played to analyze the expression change of NUPR1,HIF-1α and LASP1.Results:1.RT-q PCR revealed that lnc RNA FAL1 was highly expressed in colorectal cancer tissues(P<0.01)and five cell lines include HCT116,HT29,SW620,SW480 and Lo Vo,especially in HCT116(P<0.001)and HT29(P<0.01)cell lines.The expression level of lnc RNA FAL1 was not related to gender,age and pathological type,but was related to tumor size,TNM stage and lymph node metastasis(P<0.05).And the survival rate of colorectal cancer patients with high expression of lnc RNA FAL1 was significantly lower than that of patients with low expression(P<0.05).2.RT-q PCR results indicated that pc DNA3-lnc RNA FAL1 significantly increased lnc RNA FAL1 in HCT116 and HT29 cells(P<0.001),and pshR-lncRNA FAL1 also silenced lnc RNA FAL1 with high efficiency(P<0.01).MTT results showed that the proliferation activity of pc DNA3-lnc RNA FAL1 group was significantly higher than that of control group at 48 h after cells inoculation(P<0.05),which increased gradually with time went(72h,96h),The cell proliferation activity of psh R-lnc RNA FAL1 group was significantly decreased(P<0.05)and continued to decrease(72h,96h).The results of colony formation experiments showed that overexpression of lnc RNA FAL1 significantly enhanced the colony forming ability of HCT116 and HT29 cells(P<0.01),and silencing of lnc RNA FAL1 inhibited colony forming ability(P<0.01).Invasion and migration experiments revealed that overloaded lnc RNA FAL1 significantly increased the invasion and migration of HCT116 and HT29 cells(P<0.01),and down-regulated lnc RNA FAL1 resulted the reduction of invasion and migration cells(P<0.01).RT-q PCR and western blot results showed that up-regulation of lnc RNA FAL1 promoted EMT progression,and vice versa(P<0.01).3.Reg RNA website indicated that lnc RNA FAL1 has a binding site of mi R-637 at positions 122-144.It is speculated that lnc RNA FAL1 may be a target of mi R-637.The results of dual luciferase reporter assay showed that luciferase activity was significantly increased in p GL/Luc-lnc RNA FAL1 WT and ASO-mi R-637co-transfected group(P<0.001),while luciferase activity of p GL/Luc-lnc RNA FAL1 WT and mi R-637 mimics co-transfected group was significantly decreased(P<0.001).Overexpressed and silenced of mi R-637 resulted in low expression and high expression of lnc RNA FAL1,and vice versa,which confirmed the direct targeting relationship between the two.RT-q PCR results suggested that mi R-637 was significantly negatively expressed in colorectal cancer tissues(P<0.01),and there was a significant negative correlation between lnc RNA FAL1 and mi R-637 expression levels(R=-0.44,P <0.05).MTT assay,colony forming assay and transwell assay showed that up-regulation or down-regulation of mi R-637 might lead to decrease or enhance the abilities of proliferation,colony-forming ability,invasion and migration ability of HCT116 and HT29 cells.4.Target Scan 7.1 search results suggested that NUPR1 was a potential target gene for mi R-637.The dual luciferase reporter assay and RT-q PCR demonstrated that NUPR1 was a direct target gene of mi R-637.At the same time,RT-q PCR results showed that NUPR1 was highly expressed in cancer tissues(P<0.01),and negatively correlated with mi R-637 expression(R=-0.37,P<0.01).MTT assay,colony forming assay and transwell assay showed that down-regulation or up-regulation of NUPR1 might lead to decrease or enhance the abilities of proliferation,colony formation,invasion and migration(P< 0.001).5.Through literature search,it was found that NUPR1 affect the expression of HIF-1α,and the expression of HIF-1α and LASP1 was correlated.RT-q PCR results confirmed that knockdown or up-regulation of NUPR1 can effectively down-regulate or up-regulate the expression levels of HIF-1α and LASP1.Silencing HIF-1αeffectively reversed the effect of NUPR1 on proliferation,invasion and migration of HCT116 and HT29 cells(P<0.01).6.Overexpression of mi R-637 reversed the effect of lnc RNA FAL1 on the proliferation,invasion and migration of HCT116 and HT29 cells(P<0.01).RT-q PCR and western blot confirmed that lnc RNA FAL1 could significantly up-regulate the levels of NUPR1,HIF-1α and LASP1.To some extent,mi R-637 might reversed the effect of lnc RNA FAL1 in colorectal cancer cells(P<0.001).Conclusion:Lnc RNA FAL1 is highly expressed in colorectal cancer,and is associated with poor prognosis of coloretal cancer.Lnc RNA FAL1 plays an oncogenic effect in colorectal cancer and may significantly enhance the ability of proliferation,EMT,invasion and migration of tumor cells.Lnc RNA FAL1 promotes carcinogenesis mainly via targeting the mi R-637/NUPR1 pathway in colorectal cancer,which has certain value in the clinical diagnosis and treatment of colorectal cancer.