Long Non-coding RNA LWTA Regulates Proliferation And Migration Of Colorectal Cancer Cells

Background Colorectal cancer（CRC）is the third largest malignant tumor in the world.At present,the main treatment method is surgery combined with radiotherapy and chemotherapy.With the increase of drug tolerance and recurrence rate,the survival time of colorectal cancer patients has not improved significantly.Therefore,finding new and effective therapeutic targets and regimens is of great significance for improving patient prognosis.Long non-coding RNAs are a class of non-coding RNAs that are more than 200 nucleotides in length and cannot be translated into proteins.They are mainly involved in the occurrence and development of various malignant tumors through epigenetic regulation,transcriptional regulation and post-transcriptional regulation.The previous study of this subject found that long non-coding RNA LWTA is involved in the proliferation and migration of colorectal cancer cells,but the mechanism of action is still unclear.This study aimed to explore the mechanism of long non-coding RNA LWTA in colorectal cancer,and provide a theoretical basis for finding new potential targets for colorectal cancer therapy.Objective Screening of long non-coding RNAs（lncRNAs）related to the evolution of colorectal cancer,to clarify the molecular mechanism of lncRNA LWTA regulating the occurrence and development of colorectal cancer,and to provide new ideas for clinical targeted therapy of colorectal cancer and improvement of patient prognosis.Methods1.Collect clinical colorectal cancer and adjacent normal tissues,sequence and analyze lncRNAs that are abnormally expressed in colorectal cancer.2.Using si RNA to detect the effects of three lncRNAs with large differences on the proliferation and migration of colorectal cancer cells.3.Fluorescence in situ hybridization（FISH）was used to detect the intracellular localization of the screened lncRNAs.4.RNA pull-down and mass spectrometry identified lncRNA translation ability and binding protein.5.Immunohistochemical detection of the expression of lncRNA-encoded polype ptides in colorectal cancer and its relationship with the prognosis of patients.6.CCK8 and plate cloning experiments were used to detect the effect of the encoded polypeptide on cell proliferation.7.Transwell migration assay to detect the effect of the encoded polypeptide on cell migration.8.Prediction of the intracellular binding protein encoding the polypeptide and co-immunoprecipitation（Co-IP）to validate the prediction.Results1.Screened out 485 differential lncRNAs,among which 255 lncRNAs were down-regulated and 230 lncRNAs were up-regulated in colorectal cancer tissues.2.After si RNA interference of three lncRNAs with large differences,only lncRNA NONHSAT184602.1 could simultaneously induce changes in the proliferation and migration-related genes of colorectal cancer cells.3.NONHSAT184602.1 was mainly distributed in cytoplasm in in vitro cell culture and colorectal cancer tissue.4.NONHSAT184602.1 contains 7 open reading frames（ORF1-7）,of which ORF3 is the most expressed and can encode polypeptides.We named NONHSAT184602.1 as a long non-coding RNA with translation abilit（LWTA）.5.The polypeptide encoded by ORF3 is highly expressed in colorectal cancer tissues and is associated with poor prognosis of patients.6.CCK8 proliferation assay and plate cloning assay showed that high expression of ORF3 can significantly increase the proliferation rate of cells.7.Transwell migration assay showed that high expression of ORF3 could significantly increase the migration ability of colorectal cancer cells.8.The results of database analysis and Co-IP verification showed that the ORF3-encoded polypeptide can form a complex with RAB11 A and 14-3-3γ proteins,and we named the ORF3-encoded polypeptide as RAB11 A and 14-3-3γ scaffold protein（SPRY）.Conclusions Lnc RNA NONHSAT184602.1（LWTA）is highly expressed in colorectal cancer tissues and promotes cell proliferation and migration.LWTA mainly encodes the polypeptide SPRY and combines RAB11 A and 14-3-3γ proteins to play its role in regulating the biological functions of colorectal cancer cells.