ABSTRACT
ObjectiveTo investigate the effect of ankyrin-repeat domain-containing protein 22 （ANKRD22） on the proliferation， invasion， and migration of human hepatoma cells and its molecular mechanism. MethodsThe TCGA database was used to analyze the expression level of ANKRD22 in normal liver tissue and hepatocellular carcinoma tissue and its association with prognosis. Western Blot and qRT-PCR were used to measure the expression of ANKRD22 in human normal liver cells （L-02） and human hepatoma cells （Huh7， HepG2， MHCC-97H， SK-HEP-1， and SMMC-7721）； CCK-8 assay， EdU， wound healing assay， and Transwell assay were used to observe the effect of ANKRD22 on the proliferation， invasion， and migration of hepatoma cells； Western Blot was used to investigate the association of ANKRD22 with cyclins and EMT-related proteins； KEGG and ssGSEA analyses were performed to investigate the mechanism of action of ANKRD22 in hepatoma cells， and related experiments were conducted for validation. The independent-samples t-test was used for comparison of continuous data between two groups； a one-way analysis of variance was used for comparison between multiple groups， and the least significant difference t-test was used for further comparison between two groups. ResultsIn the TCGA database， the expression level of ANKRD22 in hepatoma tissue was significantly higher than that in normal liver tissue （t=5.083， P<0.05）， and the patients with a high expression level of ANKRD22 had longer overall survival and disease-related survival than those with a low expression level of ANKRD22 （P<0.05）. The expression level of ANKRD22 in various human hepatoma cell lines was higher than that in human normal liver cells （all P<0.05）. Cell proliferation assay showed that the ANKRD22 overexpression group had significantly higher EdU positive rate and proliferation rate than the Vector group （t=19.60 and 6.72， both P<0.001）， and compared with the si-NC group， the si-ANKRD22#2 group and the si-ANKRD22#3 group had significantly lower EdU positive rate and proliferation rate （all P<0.001）. Compared with the Vector group， the overexpression group had significantly higher expression levels of Cyclin E1， Cyclin D1， CDK7， and CDK4 （t=3.54， 4.95， 6.34， and 5.19， all P<0.01）， and the si-ANKRD22#2 group and the si-ANKRD22#3 group had significantly lower expression levels than the si-NC group （all P<0.001）. The overexpression group had a significantly lower expression level of P27 than the Vector group （t=6.12， P<0.001）， and the si-ANKRD22#2 group and the si-ANKRD22#3 group had a significantly higher expression level than the si-NC group （both P<0.001）. Invasion and migration experiments showed that compared with the Vector group， the ANKRD22 overexpression group had significantly higher migration rate and number of crossings through the membrane （migration group and invasion group） （t=5.01， 25.60， and 3.67， all P<0.05）， and compared with the si-NC group， thesi-ANKRD22#2 group and the si-ANKRD22#3 group had significantly lower migration rate and number of crossings through the membrane （migration group and invasion group） （all P<0.01）. The overexpression group had significantly higher expression levels of N-cadherin， Vimentin， and Snail than the Vector group （t=12.13， 8.85， and 13.97， all P<0.001）， and the si-ANKRD22#2 group and the si-ANKRD22#3 group had significantly lower expression levels than the si-NC group （all P<0.001）； the overexpression group had a significantly lower expression level of E-cadherin than the Vector group （t=4.98， P<0.01）， and the si-ANKRD22#2 group and the si-ANKRD22#3 group had a significantly higher expression level than the si-NC group （both P<0.001）. The KEGG enrichment analysis and the ssGSEA analysis showed that ANKRD22 was associated with the PI3K/AKT/mTOR signaling pathway in hepatocellular carcinoma， and the overexpression group had significantly higher expression levels of p-AKT/AKT， p-PI3K/PI3K， and p-mTOR/mTOR than the Vector group （t=12.21， 3.43， and 9.75， all P<0.01）， and the si-ANKRD22#2 group and the si-ANKRD22#3 group had significantly lower expression levels than the si-NC group （all P<0.001）. ConclusionANKRD22 is highly expressed in hepatoma cells and can promote the proliferation， invasion， and migration of hepatoma cells and the activation of the PI3K/AKT/mTOR signaling pathway.
ABSTRACT
Objective To investigate the expression of microRNA-1290 in pancreatic cancer and its role in invasion and metastasis of pancreatic cancer.Methods The expression of microRNA-1290 in pancreatic cancer tissue microarray and pancreatic cancer cell lines (AsPC-1,BxPC-3,Capan-2,Panc-1,and MIA PaCa-2) were detected by immunohistochemistry and QT-PCR.The pancreatic cancer cell lines Panc-1 and MIA PaCa-2 in logarithmic growth phase were treated with microRNA-1290 inhibitor,and the invasion and metastasis ability of pancreatic cancer cells were detected by Transwell and wound healing asssay.Western Blot was used to detect the expression of invasion and metastasis-associated proteins cyclooxygenase 2 (COX-2) and matrix metalloproteinase 2(MMP-2) in pancreatic cancer cell lines.Results (1) The expression of microRNA-1290 in pancreatic cancer tissues was significantly higher than that in normal pancreatic tissues and adjacent tissues (P ＜ 0.05).(2) Compared with pancreatic normal epithelial cells (HPDE),the expression of microRNA-1290 was significantly higher in different pancreatic cancer cell lines (P ＜ 0.05).The expression level of MicroRNA-1290 in Panc-1 and MIAPaCa-2 pancreatic cancer cells was significantly higher than that in other pancreatic cancer cell lines (P ＜ 0.05).(3) The number of invasive and metastatic cells was significantly decreased after treatment with microRNA-1290 inhibitor (P ＜0.05).(4) The expression of MMP-2 and COX-2 were decreased in Panc-1 and MIAPaCa-2 pancreatic cancer cells treated with MicroRNA-1290 inhibitor.Conclusion The expression of MMP-2 and COX-2 may be involved in the invasion and metastasis of pancreatic cancer cell by regulating the expression of microRNA-1290 in pancreatic cancer.