ABSTRACT
Objective To investigate the effect of miRNA-30a-3p on the proliferation,invasion and metastasis of liver cancer cells by targeting Atg3-mediated autophagy pathway.Methods The immunohistochemical staining was used to detect content of miRNA-30a-3p and Atg3 and their correlation in human hepatocellular carcinoma.Liver cancer cells were cultured in vitro and hunger environment was used to induce autophagy.RFP-GFP-LC3 double-labeled adenovirus infected hepatoma cells were used to detect autophagosomes in hepatoma cells.The expressions of autophagy-related proteins (autophagocytosis associated protein (Atg3),polyubiquitin-binding protein p62,autophagy microtubule-associated protein light chain 3 (LC3)) and EMT-related proteins (N-cadherin,vimentin,snail,ZO-1) were detected by Western blot.Platelet cloning assay and transwell assay were carried out to detect the proliferation,invasion and metastasis of carcinoma cell.CCK-8 kit was used to detect hepatocarcinoma cells' viability.Results The expression of miRNA-30a-3p was down-regulated.The expression of Atg3,E-cadherin and N-cadherin in miRNA-30a-3p high-expressed hepatocellular carcinoma was lower than that in miRNA-30a-3p low-expressed hepatocellular carcinoma.Increasing the expression of miRNA-30a-3p in hepatocellular carcinoma cells can decrease the expression of Atg3 and LC3,increase the expression of p62 and inhibit the formation of autophagosomes;otherwise,Atg3 and LC3 were increased,p62 was decreased and the formation of autophagosomes was promoted.Inhibition of Atg3 expression could decrease the expression of EMT-related proteins.When miRNA-30a-3p was inhibited,the cell viability of HCC was increased at each time point (F1 =10.314,P ＜0.05).When miRNA-30a-3p and Atg3 were inhibitor together,the cell viability of HCC was decreased at each time point(F2 =6.599,P ＜ 0.05).Conclusion miRNA-30a-3p can inhibit Atg3-mediated autophagy pathway and reduce cell autophagy activity,thus inhibiting the proliferation,invasion and metastasis of hepatocarcinoma cells.
ABSTRACT
Objective To study the expression of long non-coding RNA (lncRNA)-urothelial carcinoma associated 1 (UCA1) and miR-34b in bladder cancer and its correlation to the clinicopathologic features of bladder cancer.Methods Between January 2011 and October 2012,the expression of UCA1 and miR-34b in 5 bladder cancer cell lines (T24,BIU-87,EJ,T24-MMC,T24-ADM) and 1 normal bladder cell lines (SV-HUC-1) were measured by real-time reverse transcription-polymerase chain reaction (RTPCR).Meanwhile,the 56 bladder cancer specimens and paraneoplastic normal bladder tissues,which diagnosed by pathology were collected from bladder cancer patients undergoing radical resection of bladder.Among them,41 cases were male and 15 cases were female.The mean age was (68.4 ± 7.5)years old,range 52 to 78 years.43 cases were older than 65 years old,and 13 cases were less than 65 years old.The pathological classification included non muscle-invasive bladder cancer (NMIBC) 18 cases,muscle-invasive bladder cancer 38 cases;low grade papillary urothelial carcinoma 22 cases,high grade papillary urothelial carcinoma 34 cases;12 cases were primary lesion,the other 44 cases were diagnosed as tumor recurrence.Real-time RT-PCR was performed to analyze the expression of UCA1 and miR-34b.Results The relative expression levels of UCA1 in the normal bladder cell lines (SV-HUC-1) and 5 bladder cancer cell lines (T24,BIU-87,EJ,T24-MMC and T24-ADM) were (0.0675 ± 0.0133),(0.2934 ± 0.0531),(0.4246 ± 0.0650),(0.4206 ± 0.0826),(0.6472 ± 0.0875) and (0.7165 ± 0.1032),respectively (P ＜ 0.05).Moreover,the expression levels of UCA1 were up-regulated in 2 drug resistant bladder cancer cells lines T24-MMC (0.6472 ± 0.0875)and T24-ADM (0.7165 ± 0.1032),as compared with the T24 bladder cancer lines (0.2934 ± 0.0531),respectively (P ＜ 0.05).However,the expression levels of miR-34b in 5 bladder cancer cell lines [T24 (0.1600 ± 0.0455),BIU-87 (0.1720 ± 0.0658),EJ (0.1150 ± 0.0352),T24-MMC(0.0576 ± 0.0087),T24-ADM (0.0510 ± 0.0125)] were decreased (P ＜ 0.05),as compared with normal bladder cell lines SV-HUC-1 (0.6384 ± 0.1083).Moreover,the expression levels of miR-34b were down-regulated in 2 drug resistant bladder cancer cells lines T24-MMC (0.0576 ± 0.0087) and T24-ADM(0.0510 ± 0.0125),as compared with the T24 bladder cancer lines T24 (0.1600 ± 0.0455),respectively (P ＜ 0.05).The relative expression levels of UCA1 and miR-34b in bladder cancer tissues and paraneoplastic normal bladder tissues were (0.4225 ± 0.0714) vs.(0.0532 ± 0.0192) and (0.0340 ± 0.0134)vs.(0.5643 ±0.0616),respectively (P ＜0.05).Statistical correlation analysis showed that UCA1 to be significantly negative correlated with miR-34b in bladder cancer specimens(r =-0.54,P ＜ 0.05).The high level of UCA1 and low level of miR-34b were significantly correlated with tumor malignant grade,invasiveness and recurrence.The 3-year overall survival rate (OS) in UCA1 (+)/miR-34b(-) group (27.6％) were significantly worse compared with non UCA1 (+)/miR-34b (-) group (73.7％).Conclusion High expression of UCA1 and low expression of miR-34b were associated with the occurrence and development of bladder cancer.