Study Of Expression And Functional Of Long Non-coding RNA NORAD In Colorectal Cancer

Background and objectiveColorectal cancer is one of the most common malignant tumor of digestive system.It has the characteristics of high morbidity and high mortality in the world and It has become the third digestive tumor in China.At present,surgery is still the main treatment,and radiotherapy and chemotherapy is a complementary treatment to kill and reduce tumor cells to achieve therapeutic effects.However,the high rate of recrudesce and high drug resistance induced that 5 years of survival rate is still very low,which shows that early diagnosis and early treatment of colorectal cancer is particularly important.Therefore,it is great practical significance and theoretical value to study the occurrence and development of colorectal cancer at the molecular levels.Long non-coding RNA(lncRNA)is a non coding RNA with more than 200 nucleotides in length.In human,IncRNA is widely distributed in the genome.,although IncRNA don't encode protein,they participate in complex and important gene regulatory network sand regulate gene expression subtly.Recent studies have shown that IncRNA plays an important role in the process of normal tissue development and regulation of cell differentiation.In addition,lncRNAs is involved in the control of a variety of molecular pathways,causing changes in gene expression and regulating cells proliferation,apoptosis and cells migration.Therefore,the expression of lncRNAs is closely related to many human diseases,such as tumorigenesis.The results of human genome sequencing showed that only less than 2%of the entire genome sequence is used to encode protein and the vast majority of non-encoding sequences are transcribed into length of larger than 200 bp long noncoding RNA(lncRNA).But recent studies have shown that the expression of IncRNA in epigenetics and the level of transcription and translation regulation of oncogenes and tumor suppressor genes are involved in biological processes of cell proliferation,migration and invasion,and then influence the occurrence and development of tumor,which is a potential target for tumor diagnosis and prognosis.LncRNA NORAD is also known as LINC00657,which locate in the 20:36045622-36050960 chromosome with 5339 bp and an exon.At present,there are a few of reports about NORAD,and related bioinformatics database suggested that abnormal expression of NORAD in some tumors,which suggest that lncRNA NORAD may play an important role in the process of tumor development.Our study process comprehensive analysis of the expression and biological role of IncRNA NORAD in colorectal cancer and preliminary study of its molecular mechanism of tumor regulation firstly.This study includes three parts:the first part:the analysis of the correlation between abnormal expression of IncRNA and clinicopathological characteristics in colorectal cancer tissue;the second part:effects of regulation of IncRNA NORAD expression on migration,proliferation and invasion of colorectal cancer HT-29 cells;the last part:preliminary study on mechanism of IncRNA NORAD.Part One analysis of abnormal expression of IncRNA and correlation with clinicopathological characteristics in colorectal carcinoma tissue.Method1 LncRNA chip was used to detect the expression of IncRNA in 6 samples of colorectal cancer and corresponding adjacent normal tissue samples.2 RT-qPCR was used to detect the expression levels of 4 lncRNAs(NORAD,linc01154,nc-KRT7-2 and lnc-ITGA5-1)in 50 specimens and verify the reliability of the results of IncRNA microarray from the tissue level.3 We use RT-qPCR to detect expression of IncRNA NORAD in the 3 colorectal cancer cell lines(THC-8307,HT-29,HCT116)and a normal human intestinal epithelial cell line FHC and to validate the reliability of lncRNA chip results from the cells.4 According to the expression level of lncRNA,bivariate correlation was used to analysis relationship between IncRNA NORAD and in colorectal cancer patients gender,age and pathological type,differentiation degree,TNM stage,lymph node metastasis and distant metastasis.Result1 Colorectal cancer tissues were screened for 35 differentially expressed lncRNA,in which 20(57.14%)lncRNAs are upregulation and 15(42.86%)are downregulation,and IncRNA NORAD are significantly overexpressed in colorectal cancer tissue.2 The RT-qPCR results of 50 cases of colorectal cancer tissues and corresponding adjacent normal tissues showed that IncRNA NORAD,linc01154,nc-KRT7-2 expression in colorectal cancer tissues was significantly higher than that in adjacent normal tissues(p<0.05)and the expression of lnc-ITGA5-1 in colorectal cancer was significantly lower than that in adjacent normal tissues(p<0.05),which indicated that results of RT-qPCR was well consistent with lncRNA chip and confirmed the reliability of microarray data.3 The expression IncRNA of NORAD in colorectal cancer cell lines THC-8307,HT-29 and HCT116 was significantly higher than normal human intestinal epithelial cell lines(p<0.05),which further indicated that the expression level of NORAD was significantly increased in colorectal cancer.4 Analysis of bivariate correlation showed that expression of lncRNA NORAD significantly correlated with histological differentiation,lymph node metastasis and distant metastasis(p<0.05)and don't correlated significantly with age,gender,pathological type and tumor TNM stages(p>0.05).Part two Effects of IncRNA NORAD on proliferation,migration and invasion of colorectal cancer cell line HT-29Method1 We constructed lentiviral vector which downregulate IncRNA NORAD expression and transfected it into HT-29 cells to obtain HT-29 cells with silenced expression of IncRNA NORAD.2 CCK-8 experiment and colony-forming unit assay was used to detect the effect of silenced IncRNA NORAD on the proliferation of HT-29 cells.3 Adhesion assay were used to detect the effect of IncRNA NORAD on the adhesion ability of HT-29 cells.4 The effect of IncRNA NORAD on cell migration and invasion of HT-29 cells were detected by scratch assay and transwell assay.Result1 The pLVTHM-lncRNA NORAD vector constructed successfully,sequencing and BLAST results showed identical,RT-qPCR results showed that the expression of lncRNA NORAD in groups transfected with IncRNA NORAD 1 and IncRNA NORAD2 in HT-29 cells significantly decreased compared with the control groups(p<0.05).Then we got a HT-29 cell line with IncRNA NORAD silenced.2 Results of CCK8 experiments showed that the proliferation of SRC cells groups had no significant change at each time point after transfection(p>0.05),and lncRNA NORAD 1 groups and lncRNA NORAD2 groups of HT-29 cells appeared inhibition after transfection 12h(p<0.05)compared with the control groups,suppression increasing with time passes.3 Colony-forming experiment showed that the number of colony-forming units of SRC groups(52.4+7.4)had no significant difference(p>0.05),and the number of colony-forming units in NORAD1 groups(20.1+3.9)and NORAD2 groups(23.3+7.3)the number of clones was significantly decreased(p<0.05)compared with control groups(56.5+10.7).the results showed that when lncRNA NORAD silenced,the colony-forming ability of HT-29 cells was significantly inhibited.4 Adhesion experiments showed that:the adhesion rate of SCR groups(44.13 +5.69)have no significant difference(p>0.05),and the adhesion rate of NORAD1 groups(25.12+4.48)and NORAD2 groups(25.01+6.72)was significantly decreased(p<0.05)compared to the control groups(45.13+6.77).These results showed that silencing of lncRNA NORAD induced adhesion ability of HT-29 cells significantly decrease.5 The results of scratch assays showed that compared with the control groups,the number of migrated cells of SCR groups had no significant difference(p>0.05),and the number of migrated cells of NORAD 1 groups and NORAD2 groups was significantly decreased(p<0.05).these residue showed that the silencing of IncRNA NORAD induced migration ability of HT-29 cells was significantly inhibited.6 Transwell assays showed that compared with the control groups(56.5+10.7)compared with,the transmembrane cells number of SCR groups(52.4+7.4)have no significant difference(p>0.05),and transmembrane cells number of NORAD1 groups(20.1+3.9)and NORAD2 groups(23.3+7.3)was significantly decreased(p<0.05).These results showed that the silencing of lncRNA NORAD induced the invasion of HT-29 cells were significantly inhibited.Part Three Preliminary molecular mechanism of IncRNA NORADMethod1 Bioinformation predict target genes which interact with NORAD.2 RT-qPCR technology is used to detect the expression of ETS-1 in colorectal cancer tissue and then analyze the correlation between the expression of ETS-1 and clinical pathological characteristics.3 The lentiviral vector,which induce downregulation of ETS-1,was transfected into HT-29 cells and then to detect the effects on proliferation,migration and invasion of colorectal cancer cell line HT-29 cells by CCK-8 assay and colony-forming assay,adhesion assay and Transwell invasion assay.4 Western-blot was used to detect the effects of NORAD downregulation on the expression of uPA and MMP9 in colorectal cancer cell line HT-29 cells.5 Western-blot was used to detect the effects of ETS-1 downregulation on the expression of uPA and MMP9 in colorectal cancer cell line HT-29 cells.Result1 Compared with adjacent normal tissue,the expression of ETS1 was significantly increased(p<0.05)in colorectal cancer tissues.Statistical analysis showed that the expression of ETS1 in colorectal cancer tissue were significantly related with colorectal cancer tissue differentiation,lymph node metastasis,TNM stage and distant metastasis,and have no correlation with the patient's age,gender and histological types(p<0.05).There was positive correlation between IncRNA NORAD and ETS-1 expression in colorectal cancer tissues(R2=0.512),and they showed abnormally high expression.2 Downregulated the expression of ETS-1 in colorectal cancer cell HT-29 inhibited cell growth and clone formation,adhesion,migration and invasion of colorectal cancer HT-29 cells.3 Downregulated the expression of ETS-1 in colorectal cancer cell significantly inhibited the expression level of uPA and MMP9 in HT-29 cells.4 Downregulated the expression of IncRNA NORAD significantly reduced the expression of ETS-1,MMP9 and uPA in colorectal cancer HT-29 cells.Conclusion1 35 differentially expressed IncRNAs were screened in colorectal cancer,among them 20(57.14%)lncRNAs upregulating and 15(42.86%)downregulating.Abnormally high expression of IncRNA NORAD and ETS1 is the positive correlation in expression,and the degree of differentiation,lymph node metastasis and distant metastasis were significantly related in colorectal cancer tissue,2 Downregulated the expression of NORAD lncRNA effectively inhibit the proliferation,migration and invasion of colorectal cancer HT-29 cells.3 LncRNA NORAD may regulate the expression of MMP9 and uPA by affecting the expression of ETS-1 gene and play a role in promoting the proliferation,migration and invasion of colorectal cancer cells.