Effect of miR-30c/YWHAZ axis on drug resistance of pancreatic cancer cells / 中华内分泌外科杂志

Objective:To investigate whether microRNA-30c (miR-30c) mediates the resistance of pancreatic cancer cells to gemcitabine (Gb) by targeting tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activating protein zeta polypeptide (YWHAZ) .Methods:SW1990 cell line with the lowest expression of miR-30c in human pancreatic cancer cell lines was screened by RT-qPCR. Gb-resistant cell line SW1990/Gb was established and divided into SW1990/Gb group (untransfected) , miR-30c over expression (Ad-miR-30c) group, Ad-miR-30c negative control (Ad-eGFP) group, and SW1990 group. The level of miR-30c was measured by RT-qPCR; the half inhibitory concentration (IC50) and drug resistance index (IR) were measured by CCK-8 method; the expression of drug resistance-related protein P-gp, apoptosis-related protein Caspase-1, migration and transfer-related proteins MMP-9, YWHAZ and downstream pathway-kinase mitogen-activated protein kinase (p38MAPK) /extracellular regulatory protein kinase 1 (ERK1) protein was measured by Western blot. After co-transfection of Ad-miR-30c and YWHAZ overexpressing adenovirus (Ad-YWHAZ) , the expression of P-gp and YWHAZ pathway related proteins was measured by Western blot method.Results:The IC50 (59.16±5.14, nmol/L) , IR (11.15±0.19) , expressions of YWHAZ protein (1.59±0.15) and P-gp (2.43±0.26) in SW1990/Gb-resistant cells were high, the expression of miR-30c (0.25 ±0.02) was low ( P<0.05) , and the p38MAPK/ERK pathway was activated. After up-regulating the expression of miR-30c (1.59±0.15) in SW1990/Gb cells, the IC50 (25.14±2.15, nmol/L) and IR (5.48±0.12) , YWHAZ (1.49±0.13) , P-gp (1.46± 0.10) decreased ( P<0.05) , and the p38MAPK/ERK pathway was activated. Up-regulating the expression of YWHAZ could reverse the above-mentioned effects of Ad-miR-30c ( P<0.05) . Conclusions:The expression of miR-30c is low in pancreatic cancer Gb-resistant cell lines. Up-regulating the expression of miR-30c can target and inhibit the YWHAZ/p38MAPK/ERK pathway, inhibit the expression of drug-resistant protein P-gp, and reduce the resistance of pancreatic cancer cells to Gb.

Mechanism of miR-30c over-expression inhibiting malignant phenotypes of cervical cancer cells / 中国病理生理杂志

AIM:To investigate the molecule mechanism of microRNA(miR)-30c over-expression inhibiting malignant phenotypes of cervical cancer cells.METHODS:Cervical cancer cell lines C33A,HeLa,SiHa and CaSki were transfected with pGenesil-1-miR-30c plasmid using Lipofectamine 2000 kit,and the expression of miR-30c was determined by TaqMan real-time PCR.The cell viability inhibition rate,colony formation ability,migration rate and apoptotic rate were measured by MTT assay,colony formation assay,Transwell experiment,and flow cytometry with Annexin V-FITC staining. The protein expression of Bax,Bcl-2, matrix metalloproteinase(MMP)-9, MMP-13 and tissue inhibitor of metalloprotei-nase-1(TIMP-1)was detected by Western blot.RESULTS:The expression levels of miRNA-30c in the cervical cancer cell lines transfected with pGenesil-1-miR-30c plasmid were significantly higher than those in negative control groups(cell lines transfected with pGenesil-1 plasmid)(P<0.01).Significantly increased cell viability inhibition rate,and decreased colony formation ability and migration rate were found in the cervical cancer cell lines over -expressing miR-30c as compared with negative control groups(P<0.05).The apoptotic rate in the cervical cancer cell lines over-expressing miR-30c was dramatically higher than that in control groups(P<0.05).Over-expression of miR-30c in cervical cancer cells promoted the protein expression of Bax and TIMP-1,and decreased the protein expression of Bcl-2 and MMP-13(P<0.05 or P<0.01).CONCLUSION:Over-expression of miR-30c significantly inhibits the viability and migration,and induces apop-tosis of cervical cancer cells.The mechanism may be related to activating apoptosis pathway and inhibiting MMP-13 protein expression.

Serum microRNA-30c levels are correlated with disease progression in Xinjiang Uygur patients with chronic hepatitis B

We aimed to investigate the potential role and mechanism of microRNA-30c (miR-30c) in the pathological development of chronic hepatitis B (CHB). The serum levels of miR-30c in hepatitis B virus (HBV) carrier Xinjiang Uygur patients with inactive, low-replicative, high-replicative and HBe antigen-positive CHB were investigated. HepG2 cells were co-transfected with pHBV1.3 and miR-30c mimic or inhibitor or scramble RNA. The effects of miR-30c dysregulation on HBV replication and gene expression, cell proliferation and cell cycle were then investigated. miR-30c was down-regulated in Xinjiang Uygur patients with CHB compared to healthy controls and its expression level discriminated HBV carrier patients with inactive, low-replicative, high-replicative and HBe antigen-positive risk for disease progression. Overexpression of miR-30c significantly inhibited HBV replication and the expressions of HBV pgRNA, capsid-associated virus DNA and Hbx in hepatoma cells. Moreover, overexpression of miR-30c significantly inhibited cell proliferation and delayed G1/S phase transition in hepatoma cells. Opposite effects were obtained after suppression of miR-30c. Our results indicate that miR-30c was down-regulated in Xinjiang Uygur patients with CHB, and miR-30c levels could serve as a marker for risk stratification of HBV infection. Down-regulation of miR-30c may result in the progression of CHB via promoting HBV replication and cell proliferation.

Effects of miR-30 c on viability and migratory ability of HUVECs by tar-geting PAI-1 / 中国病理生理杂志

AIM: To investigate the effect of microRNA (miR)-30c on the viability and migratory ability of human umbilical vein endothelial cells (HUVECs) by targeting plasminogen activator inhibitor-1 (PAI-1).METHODS:The HUVECs were transfected with miR-30c mimic and inhibitor or negative control (NC), and then the expression levels of miR-30c, PAI-1 mRNA and protein were detected by RT-qPCR and Western blot.The viability and migratory ability of HUVECs were measured by CCK-8 assay and wound healing test .After bioinformatic analysis, the assessment of miR-30c binding to PAI-1 3’-UTR was carried out using a luciferase reporter gene assay .RESULTS:miR-30c directly down-regu-lated PAI-1 levels by binding to the 3’ UTR seed sequence of PAI-1 mRNA.Furthermore, transfection of a miR-30c mimic down-regulated the expression of PAI-1 at mRNA and protein levels, leading to enhanced migratory ability and viability of the HUVECs.However, transfection of a miR-30c inhibitor up-regulated the expression of PAI-1 at mRNA and protein le-vels, leading to decreased migratory ability and viability .CONCLUSION:Regulation of miR-30c level changes the migra-tory ability and viability of HUVECs by affecting the PAI-1 expression, indicating the involvement of miR-30c in modulating endothelial function .