MiR-101 And MiR-26a Targeting ASPN Suppresses Tumor Proliferation And Metastasis By Regulating The PI3K/AKT Signalling In Colorectal Cancer

Objective To investigate the clinical significance of ASPN expression in colorectal cancer and its predictive value for the survival of colorectal cancer(CRC)patients.To explore the effect of ASPN on invasion and metastasis of colorectal cancer cells.To find the miRNAs targeting ASPN,and to study the relationship between them.Finally,the effect of miRNA-ASPN on the signal pathway of CRC was discussed.Methods The expression levels of ASPN in 203 patients with CRC,80 patients with colorectal benign lesions and 186 healthy controls were detected by immunohistochemical staining.Western Blot was used to validate the CRC patient tissue and the CRC cell line.Select ASPN relatively high expression cell lines(Caco-2,SW-620),siASPN and its negative control were transfected into cell lines.The cell proliferation ability was detected by CCK8 assay.Transwell assay was used to detect the changes of cell invasion and migration ability Western blot was used to detect the changes of E-cadherin and vimentin protein.Using bioinformatics software to predict the role of miRNA(miR-101,mi R-26a)in the regulation of ASPN.The construction of a double luciferase reporter gene assay.The relative expression levels of miR-101 and miR-26 a in 32 cases of CRC patients and their adjacent tissues were detected by real-time fluorescence quantitative PCR.MiR-101,miR-26 a mimics and negative control were transfected into cell lines were transfected into Caco-2 and SW-620 cell lines,The changes of cell proliferation were measured by CCK8 assay.Transwell assay was used to detect the changes of cell invasion and migration ability.The changes of E-cadherin and vimentin protein were detected by Western Blot.The rescue experiment further verified the relationship between them.The levels of PI3 K and p-PI3 K were detected by Western Blot.The effect of ASPN,miR-101 and miR-26 a on the proliferation of CRC was evaluated by nude mice MiR-101,miR-26 a mimetic and miR-26 a were transfected into Caco-2 and SW-620 cell lines,and the expression of mi R-101 and miR-26 a was detected by CCK8 The changes of cell proliferation were observed by Transwell assay.The changes of E-cadherin and vimentin protein were detected by Western Blot.The cross-expression plasmids of ASPN were transfected into Caco-2 and SW-620 cell lines by miR-101 and miR-26 a mimics respectively.The relationship between PI3 K,p-PI3 K protein levels.The effect of ASPN,mi R-101 and miR-26 a on the proliferation of CRC was evaluated by nude mice tumorigenesis.Results IHC-P showed a significant increase in the positive rate of ASPN in CRC patients compared with colorectal benign lesions and healthy controls.By sorting out patient data and data analysis,high expression of ASPN is a risk factor for CRC,leading to poor prognosis and shortened survival.Knockdown of ASPN resulted in the proliferation of CRC cells,and the ability of invasion and migration was weakened,which was manifested in the down-regulation of CCK-8 proliferation curve,the change of scratch test and the change of Transwell experiment and the change of related transfer protein(E-cadherin increased and vimentin reduction).Bioinformatics software,Western Blot and double luciferase reporter gene assay co-verified that ASPN was regulated by mi R-101 and miR-26 a in CRC.The relative expression levels of miR-101 in cancer tissues and adjacent tissues of 32 patients with CRC were 0.7888 ± 0.1702 and 3.598 ± 0.8063 respectively.The relative expression levels of miR-26 a in cancer tissues and adjacent tissues of patients with CRC were 0.9989 ±0.3395 and 4.633±1.546.The expression levels of mi R-101 and miR-26 a in cancer tissues were significantly lower than those in adjacent tissues(P<0.05,0.0012 and 0.0251,respectively).The effect of mi R-101 and miR-26 a mimics on the function of CRC cells was consistent with knocking down ASPN,which also resulted in the proliferation and invasion and migration of CRC cells,which was manifested in the down-regulation of CCK-8 proliferation curve,Changes in experimental trends and changes in related transfer proteins(E-cadherin increased and vimentin reduction).The effect of mi R-101 and mi R-26 a on ASPN was confirmed by the rescue experiment.The proliferation of cells was positive and the invasion and migration ability were enhanced after transfection of ASPN plasmid,but after transfection of miR-101 and miR-26 a mimics respectively,all the above trends down.Western Blot found that the levels of p-PI3 K protein were changed,but the PI3 K protein did not change.In the nude mice,the knockdown of the ASPN group compared with the control group,long survival time,light in weight and cell proliferation ability is weak;the same performance in miR-101,miR-26 a mimics group.Conclusions In CRC,ASPN showed high expression,and indicates poor prognosis and shortened survival.Knocking down ASPN to make CRC cell proliferation,invasion and migration ability weakened.MiR-101 and miR-26 a can negatively regulate ASPN in CRC,and the relative expression in cancer tissue is lower than that in adjacent tissues,and it plays a role in the diagnosis and prognosis of CRC.MiR-101 and miR-26 a can reverse the carcinogenic effect of ASPN and inhibit the proliferation,invasion and migration of CRC cells by activating PI3K/AKT signaling pathway.