Long Noncoding RNA NR2F2-AS1 Spongs MiR-106b-5p To Inhibit Metastasis And Invasion Of Colorectal Cancer Through Upregulating PLEKHO2

Background:Colorectal cancer(CRC)is a malignant epithelial tumor with glandular characteristics that originates in the large intestine and in most cases appears as adenocarcinoma.The incidence of most colorectal cancer is sporadic and has nothing to do with genetic susceptibility or family history;however,20-30% of colorectal cancer patients have a family history of colorectal cancer and 5% of them meet the tumorigenesis and meet Mendelian genetic synthesis Sign.In China and throughout the world,the incidence and mortality of CRC has been increasing,which seriously threatens people’s health.This phenomenon is partly due to the lack of reliable biomarkers for early prevention prediction,diagnosis and prognosis of CRC.With the development of genomics and bioinformatics,a variety of biological databases have been gradually established and developed.We can screen new candidate genes for CRC through the database at zero cost.This will help in vitro functional experiments and the study of molecular mechanisms.We know the pathogenesis of CRC,so we can propose new strategies for CRC prevention,early detection and treatment,and hope to reduce the incidence and mortality of CRC.Long non-coding RNAs(lncRNAs)are non-coding RNAs which are longer than 200 nucleotides in length and may be related to a variety of known cancer genes.Increasing evidence shows that lncRNA plays an important role in tumorigenesis and cancer metastasis.It can be used as endogenous competitive RNA(ce RNAs),which spongs miRNAs through negative regulation to reduce the Inhibition of miRNAs on downstream target genes which plays an important role in the regulation of human diseases and may be a prognostic marker for many cancers including CRC.Research method:1、 First check whether there is a difference in the expression of miR-106b-5p between the normal and the cancer sample of CRC in the oncomiR database,and further inquire about its relationship with the T,N,and M stages of the CRC.Mi R-106b-5p was overexpressed in SW480 cells,and miR-106b-5p was inhibited in HCT116 cells.The overexpression and inhibition efficiency were first verified by q-PCR.The effects of miR-106b-5p on the biological behavior of SW480 and HCT116 cells were determined by cck8,clone formation,transwell migration and invasion,and apoptosis experiments to determine the role of miR-106b-5p in CRC.2、Lnc RNA NR2F2-AS1 and m RNA PLEKHO2 negatively correlated with miR-106b-5p expression were predicted from biological database databases such as DIANA,miRTar Base,Target Scan,miRDB,and TCGA;after overexpressing miR-106b-5p in SW480 cells used qPCR to verify the effect between miR-106b-5p 、 NR2F2-AS1 and PLEKHO2.Then,the Pearson correlation coefficient analysis is performed to determine whether there is a correlation between NR2F2-AS1 and PLEKHO2.3、Predicted the binding sequence between miR-106b-5p and NR2F2-AS1 nc RNA through the database,synthesize the wild-type gene fragment that miR-106b-5p combines with NR2F2-AS1 nc RNA sequence,and construct NR2F2-AS1 dual luciferase Reporter gene wild-type vector(wt-NR2F2-AS1)and mutant vector(mut-NR2F2-AS1).The dual luciferase reporter gene detection system was used to detect Regulatory effect on HCT116 cells;then transfected HCT116 cells with NR2F2-AS1 si RNAs,selected si RNAs with significant knockdown efficiency and stable si RNAs,and verified the effects of NR2F2-AS1 on proliferation and migration of HCT116 cells after transfection,and verified knockdown at the protein level in order to verify wheater there is an inhibitory effect between NR2F2-AS1 and PLEKHO2 gene expression.4、Predicted the binding sequence of miR-106b-5p to the 3’UTR region of PLEKHO2 through the database,synthesize the wild-type gene fragment that miR-106b-5p binds to the 3’UTR region of PLEKHO2,then construct a PLEKHO2 3’UTR dual luciferase report gene wild-type vector(wt-PLEKHO2 3’UTR)and mutant vector(mut-PLEKHO2 3’UTR)to tested the regulation between miR-106b-5p and PLEKHO2 by the dual luciferase reporter gene detection system;HCT116 cells were transfected with three PLEKHO2 si RNAs,found the most significant and stable si RNA knockdown efficiency was selected,after transfected,to verify the effect of PLEKHO2 on the proliferation and migration of HCT116 cells.5、Finally,verify the changes in the expression of PLEKHO2 gene at the RNA and protein level through a recovery experiment,and then verify the changes in the biological behavior of HCT116 cells through a recovery experiment to fully confirm the relationship between NR2F2-AS1,miR-106b-5p and PLEKHO2.Result:1、The expression of miR-106b-5p was significantly increased in colon and rectal cancer tissues,and was related to the T,N,and M stages of rectal cancer.Overexpression of miR-106b-5p in SW480 cells,the overexpression efficiency is more than 200 times,and overexpression of miR-106b-5p promotes the proliferation,migration and invasion of SW480 cells,and inhibits their cell apoptosis;what ’more inhibits the expression of miR-106b-5p can inhibit the proliferation,migration and invasion of HCT116 cells,and promote their apoptosis.2、The lncRNA with negative regulation effect on miR-106b-5p expression predicted by the database is NR2F2-AS1,and the m RNA is PLEKHO2;q-PCR results show that miR-106b-5p is over-expressed in SW480 cells were significantly inhibited the expressions of NR2F2-AS1 and PLEKHO2;the results of pearson correlation analysis showed that the expressions of NR2F2-AS1 and PLEKHO2 have a positive correlation.3、The results of the dual luciferase reporter gene detection system further demonstrate that miR-106b-5p can sponge to NR2F2-AS1;after HCT116 cells were transfected with NR2F2-AS1’s si RNA,the expression of NR2F2-AS1 at the RNA level was significantly reduced;The expression of PLEKHO2 at the protein level also showed a significant downward trend,indicating that NR2F2-AS1 and PLEKHO2 have a positive regulation effect;functional experiment results show that knocking down NR2F2-AS1 promotes the migration and invasion of HCT116 cells,but the impact of proliferation and apoptosis is not significant.4 、 The results of the dual luciferase reporter gene detection system confirmed that PLEKHO2 is a target-gene for miR-106b-5p.After transfection of PLEKHO2’s si RNA,the expression of PLEKHO2 at both the RNA level and the protein level was reduced,which verified that its si RNA knockdown efficiency;and knocking down PLEKHO2 will promote the migration and invasion ability of HCT116 cells,but the effects on proliferation and apoptosis are not as significant as NR2F2-AS1.5、The q-PCR results of the rescue experiments showed that the si RNA of NR2F2-AS1 and PLEKHO2 had a significant knockdown effect on the PLEKHO2’s expression,and inhibitor miR-106b-5p had a promoting effect on the expression of PLEKHO2;in addition,the addition of NR2F2-AS1 and PLEKHO2 si RNA will inhibit the expression of PLEKHO2;the protein results are highly consistent with q-PCR results;functional experiment results show that knocking down the expression of NR2F2-AS1 and PLEKHO2 will promote HCT116 cell migration and invasion ability.Inhibitor miR-106b-5p will inhibit the migration and invasion ability of HCT116 cells.However,the migration of HCT116 cells was found after transfection of inhibitor mit R-106b-5p with NR2F2-AS1 and PLEKHO2.And the ability to attack has been restored to a certain extent.Conclusion:miR-106b-5p plays an oncogene role in colorectal cancer and promotes the proliferation and migration of CRC,lncRNA NR2F2-AS1 and m RNA PLEKHO2 related to it act as tumor suppressor genes in colorectal cancer,and they can inhibit the ability of colorectal cancer cells to migrate and invade.NR2F2-AS1 can upregulate the expression of PLEKHO2 by competitively binding miR-106b-5p and inhibit the migration and invasion of colorectal cancer cells.This result suggests that abnormal expression of NR2F2-AS1 may be an early event of CRC.In the future,it may be a target gene for early screening of colorectal cancer,and it can regulate the development of CRC by regulating the expression of NR2F2-AS1.