International Journal of Molecular Medicine

Christophersen, Mikael ; Høgdall, Claus ; Høgdall, Estrid

doi: N/Apmid: N/A

Ovarian cancer is the most lethal gynaecological malignancy. The cancer initially presents with non‑specific symptoms; thus, it is typically not discovered until the patient has reached the late, considerably more lethal, stages of the disease. Research focus is currently on finding novel biomarkers, especially for early detection and stratification of the disease. One promising approach has been to focus on mutations or variations in the genetic code that are associated with the risk of developing ovarian cancer. A certain heritable component is already known regarding genes such as BRCA1/2, TP53, MSH6, BRIP1 and RAD51C, yet these are estimated to only account for ~3.1% of the total risk. Recent advances in sequencing technologies have enabled the investigation of hundreds of thousands of genetic variants in genome‑wide association studies in tens of thousands of patients, which has led to the discovery of 108 (39 loci with P<5.0x10‑8) novel susceptibility loci for ovarian cancer, presented in this review. Using the published variants in a patient cohort screening, together with variants identified in our ongoing whole exome sequencing project, future aims are to ascertain whether certain of the novel variants could be used as biomarkers for early diagnosis and/or treatment decisions.

Zhao, Fenglin ; Chen, Fuqiang ; Yuan, Xin ; Liu, Yiting ; Chen, Jie

doi: N/Apmid: N/A

Parastomal hernia (PH) is a common complication following stoma formation. Abnormal collagen synthesis has been suggested to be involved in PH. The aim of the present study is to explore the effect and mechanism of the collagen synthesis on PH. Data from 157 patients with rectal cancer who received permanent colostomy were retrospectively collected and analyzed to identify the risk factors for PH. Primary culture of skin fibroblasts from patients with or without PH were performed. Cell viability, migration and invasion levels were detected by Cell Counting Kit‑8, and wound healing and Transwell assays, respectively. Reverse transcription quantitative polymerase chain reaction and western blot analysis assays were performed to measure the gene and protein expression levels, respectively. The risk factors of sex, body mass index, aperture size and collagen expression were closely associated with the occurrence of PH. α1 (III) procollagen expression levels were significantly increased in patients with PH, while no marked difference in α1 (I) procollagen mRNA expression levels were observed in patients with or without PH. The viability and motility of fibroblasts from the patients with hernia were suppressed. The expression levels of matrix metalloproteinase (MMP)‑2 and MMP‑9 were decreased while the levels of collagen III and metalloproteinase inhibitor 1 (TIMP‑1) were increased in the fibroblasts from the patients with PH. Silencing TIMP‑1 expression promoted fibroblast migration and invasion and reversed the patterns of MMP‑2, MMP‑9 and collagen III expression in fibroblasts from the patients with PH. Decreased collagen III may inhibit the development of PH, potentially through decreases in TIMP‑1 expression. Therefore, the results from the present study may provide a novel target for PH therapy.

Li, Nan ; Wang, Li‑Juan ; Xu, Wei‑Long ; Liu , Su ; Yu, Jiang‑Yi

doi: N/Apmid: N/A

MicroRNAs (miRNAs or miRs) play an important role in pathological processes in diabetic nephropathy (DN). This study aimed to explore whether miR‑379‑5p is associated with renal fibrosis in DN and to elucidate the underlying mechanisms. In vitro experiments indicated that miR‑379‑5p expression was downregulated by high glucose (HG) treatment in mouse mesangial cells (MMCs). Transfection with miR‑379‑5p mimics suppressed the proliferation and the accumulation of extracellular matrix (ECM) components, which were promoted by HG treatment. LIN28B was proven to be a direct target of miR‑379‑5p by luciferase report assay. In addition, the loss of expression of LIN28B, as well as the decrease in cell proliferation and in the accumulation of ECM components, which were induced by the knockdown of LIN28B, were attenuated in the MMCs following transfection with miR‑379‑5p inhibitors. Furthermore, type 2 diabetic db/db mice were used to examine the efficiency of miR‑379‑5p agomir in the alleviation of renal fibrosis. Consistent with the results of the in vitro experiments, miR‑379‑5p agomir suppressed mesangial cell proliferation and the accumulation of ECM components by regulating the LIN28B/let‑7 pathway. Taken together, the findings of this study suggest that miR‑379‑5p is highly involved in renal fibrosis in DN, and that it may be a potential effective therapeutic target for DN.

Pan, Changwei; Lang, Hongxin; Zhang, Tao; Wang, Rui; Lin, Xuewen; Shi, Ping; Zhao, Feng; Pang, Xining

doi: N/Apmid: N/A

Stem cells derived from human amniotic membrane (hAM) are promising targets in regenerative medicine. A previous study focused on human amniotic stem cells in skin wound and scar‑free healing. The present study aimed to investigate whether hydrogen peroxide (H2O2)‑induced senescence of human dermal fibroblasts (hDFs) was influenced by the anti‑aging effect of conditioned medium (CdM) derived from human amniotic stem cells. First, the biological function of two types of amniotic stem cells, namely human amniotic epithelial cells (hAECs) and human amniotic mesenchymal stem cells (hAMSCs), on hDFs was compared. The results of cell proliferation and wound healing assays showed that CdM promoted cell proliferation and migration. In addition, CdM from hAECs and hAMSCs significantly promoted proliferation of senescent hDFs induced by H2O2. These results indicated that CdM protects cells from damage caused by H2O2. Treatment with CdM decreased senescence‑associated β‑galactosidase activity and improved the entry of proliferating cells into the S phase. Simultaneously, it was found that CdM increased the activity of superoxide dismutase and catalase and decreased malondialdehyde by reducing H2O2‑induced intracellular reactive oxygen species production. It was found that CdM downregulated H2O2‑stimulated 8‑hydroxydeoxyguanosine and γ‑H2AX levels and decreased the expression of the senescence‑associated proteins p21 and p16. In conclusion, the findings indicated that the paracrine effects derived from human amniotic stem cells aided delaying oxidative stress‑induced premature senescence.

Lee, Sung‑Eun ; Kwon, Tae‑Rin ; Kim, Jong ; Lee, Byung‑Chul ; Oh, Chang ; Im, Minju ; Hwang, Yu ; Paik, Sang ; Han, Seungryel ; Kim, Jeom‑Yong ; Kim, Beom

doi: N/Apmid: N/A

Zhu, Ying ; Shi, Qiong ; Peng, Qi ; Gao, Yue ; Yang, Ting ; Cheng, Yu ; Wang, Hao ; Luo, Yetao ; Huang, Ailong ; He, Tong‑Chuan ; Fan, Jiaming

doi: N/Apmid: N/A

Although a number of experimental models have been developed for liver research, each has its own advantages and disadvantages. The present study attempted to develop a simple and effective 3‑dimensional mouse liver microsphere tissue culture (LMTC) model in vitro for the analysis of hepatic functions. Hepatic characteristics and potential applications of this model were compared with that of mouse model in vivo and mouse primary hepatocytes in vitro. Using freshly‑perfused mouse liver tissue passed through 80‑mesh sift strainer (sift80), it was demonstrated that under the optimal culture conditions, the sift80 microsphere tissue cultured in 2% bovine calf serum medium remained viable with marked proliferating cell nuclear antigen and anti‑Myc proto‑oncogene protein expression, exhibited normal hepatic functions including indocyanine green (ICG) uptake/release and periodic acid‑Schiff staining, and expressed hepatocyte‑specific genes for up to 2 weeks. The microsphere tissue was responsive to bone morphogenic protein 9 (BMP9) stimulation leading to upregulation of downstream targets of BMP9 signaling. Furthermore, 3 commonly‑used liver‑damaging drugs were indicated to effectively inhibit hepatic ICG uptake, and induce the expression of hepatotoxicity‑associated genes. Therefore, this simplified LMTC model may be a useful in vitro tissue culture model to investigate drug‑induced liver injury and metabolism, and hepatocyte‑based cell singling.

Li, Qianyuan ; Zhou, Xiukou ; Fang, Zhengyu ; Zhou, Huamiao

doi: N/Apmid: N/A

Kallikrein‑related peptidase 12 (KLK12) is overexpressed in cancer tissues including gastric, breast and prostate cancer. However, the role of KLK12 in colorectal cancer is not fully understood. In the present study, the level of KLK12 was determined by performing reverse transcription‑polymerase chain reaction (RT‑qPCR) in colorectal cancer tissues and cell lines. Lipofectamine® 2000 was used to transfect HT‑29 cells to overexpress and knockdown KLK12. Cell viability, migration, invasion and apoptosis were detected by MTT, wound healing, Transwell and flow cytometry assays, respectively. The mRNA and protein expression levels of EMT‑associated proteins, apoptosis‑associated proteins, phosphorylated adenosine monophosphate‑activated protein kinase (p‑AMPK) and phosphorylated mammalian target of rapamycin (p‑mTOR) were determined by RT‑qPCR and western blot analysis. It was identified that the KLK12 mRNA levels were increased significantly in colorectal cancer tissues and cell lines. KLK12 small interfering RNA inhibited cell viability, migration and invasion. Furthermore, epithelial‑mesenchymal transition (EMT)‑associated proteins were altered by siKLK12. Cell apoptosis was induced by KLK12 downregulation, which was demonstrated by the changes in apoptosis‑associated proteins; however, KLK12 overexpression produced the opposite effect. SiKLK12 enhanced the expression of p‑AMPK and suppressed the expression of p‑mTOR, while KLK12 overexpression had the opposite effect. Promotion of KLK12 overexpression‑induced cell viability was reversed by 5‑aminoimidazole‑4‑carboxamide ribonucleotide, an activator of the AMPK signaling pathway, and rapamycin, a specific inhibitor of the mTOR signaling pathway. Taken together, the results of the present study indicated that KLK12 was overexpressed in colorectal cancer and may regulate cell behavior, potentially via the AMPK and mTOR pathways.

Bao, Chuncha ; Yang, Zhong ; Cai, Qiyan ; Li, Qian ; Li, Hongli ; Shu, Bin

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Recent studies have confirmed that kidney tissue fibrosis is closely linked to the natural aging of organs. One of its major characteristics is the reduction of autophagic activity. However, to date, few studies have assessed whether incremental load training is able to improve the occurrence of renal fibrosis caused by natural aging and the underlying mechanisms. In the present study involving male C57/BL mice, an elderly exercise group (OY group) was subjected to progressive load‑increasing rotary‑bar training (5 days/week, lasting for 6 weeks), with an elderly control group (OC group) and a young control group (YC group) used as controls. Renal fibrosis and autophagy‑associated indicators were assessed by Masson's staining, reverse transcription‑quantitative PCR analysis, western blotting, immunofluorescence and transmission electron microscopy. The results suggested that collagen deposition in the basal part of the renal tubular epithelium and glomeruli in the OY group was significantly lower than that in the OC group. In the OC group, the protein expression levels of E‑cadherin, Beclin 1 and light chain 3 were significantly decreased, and increases in α‑smooth muscle actin‑positive signals were observed in the glomerular matrix and renal capsule wall. Furthermore, the expression of transforming growth factor (TGF)‑β1 and its downstream signaling molecules TGF‑β‑activated kinase 1 (TAK1), mitogen‑activated protein kinase (MAPK) kinase (MKK3) and p38MAPK were downregulated following training. The present study confirmed that incremental load training may improve renal fibrosis in aged mice by regulating the TGF‑β1/TAK1/MMK3/p38MAPK signaling pathway and inducing the activation of autophagy to reduce the synthesis of extracellular matrix and delay the epithelial‑mesenchymal transition. The present study provides a novel experimental basis for the intervention of incremental load training to prevent senile renal fibrosis.

Chen, San‑Yuan ; Huang, Hsin‑Yi ; Lin, Han‑Pei ; Fang, Chiung‑Yao

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Biliary cancer (BC) is an aggressive neoplasm with high mortality. BC can be categorized into three groups: Intrahepatic cholangiocarcinoma (CCA; also known as bile duct cancer), extrahepatic cholangiocarcinoma and gallbladder cancer. Due to its heterogeneity and aggressiveness, the response to current chemotherapy and radiotherapy methods in patients with BC is poor. Therefore, there is an urgent requirement to develop drugs to treat BC. Piperlongumine (PL), a naturally occurring small molecule isolated from Piper longum L., exhibits anticancer activity by inducing reactive oxygen species (ROS) production. In the present study, the effects of PL on cell proliferation, cell cycle, apoptosis and autophagy in BC cells were investigated. PL induced BC cell death in a concentration‑ and time‑dependent manner by inducing ROS production. PL induced cell cycle arrest in CCA cells (HuCCT‑1) and gallbladder cancer cells (OCUG‑1) cells, but with distinct cell cycle distribution profiles. PL caused G2/M cell cycle arrest in HuCCT‑1 cells, and G0/G1 cell cycle arrest in OCUG‑1 cells. PL induced apoptosis and autophagy; PL treatment induced accumulation of LC3‑II in a concentration‑ and time‑dependent manner. The Erk signaling pathway appeared to be involved in autophagy induction. Application of the ROS scavenger, N‑acetyl‑l‑cysteine, to BC cells attenuated the cell death, cell cycle arrest, apoptosis and autophagy induced by PL treatment. These findings indicated that PL may be a potential agent for BC treatment in the future.

Xue, Dan ; Yang, Ping ; Wei, Qiongying ; Li, Xiaoping ; Lin, Lan ; Lin, Tingyan

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Lung cancer is considered to be one of the world's deadliest diseases, with non‑small cell lung cancer (NSCLC) accounting for 85% of all lung cancer cases. The present study aimed to investigate the role and underlying mechanisms of interleukin‑21 (IL‑21), and its receptor IL‑21R, in NSCLC. Lung tissues and blood samples of NSCLC were used to measure IL‑21, IL‑21R and programmed death 1 ligand 1 (PD‑L1) expression using ELISA, western blot and immunohistochemistry analyses. Following treatment with different doses of IL‑21, the proliferation, invasion and migration of human NSCLC cell line A549 was evaluated using a cell counting kit‑8, colony formation, Transwell and scratch wound healing assays, respectively. Additionally, IL‑21R and PD‑L1 expression in A549 cells was detected using western blot analysis and immunofluorescence. IL‑21R silencing was subsequently used to investigate its effects in cell proliferation, invasion and migration. PD‑L1, IL‑1β and tumor necrosis factor α (TNF‑α) expression were measured. Finally, Wnt/β‑catenin signaling expression was evaluated using western blot analysis following treatment with IL‑21. Cells were then treated with lithium chloride (LiCl), which is an agonist of Wnt/β‑catenin signaling, and the levels of PD‑L1, IL‑1β and TNF‑α were detected. The results revealed that IL‑21 and IL‑21R expression in the lung tissues and blood samples of patients with NSCLC were decreased, while PD‑L1 expression was increased, compared with normal tissues or healthy controls. Treatment of A549 cells with IL‑21 upregulated IL‑21R expression, downregulated PD‑L1 and inhibited cell growth and metastasis in a dose‑dependent manner. Following IL‑21R silencing, the effects of IL‑21 treatment were reversed, suggesting that IL‑21 acted on A549 cells through binding to IL‑21R. In addition, the results demonstrated that IL‑21 treatment reduced the expression levels of proteins associated with the Wnt/β‑catenin signaling, whereas activation of Wnt/β‑catenin signaling with the LiCl agonist upregulated PD‑L1, IL‑1β and TNF‑α expression. In conclusion, the IL‑21/IL‑21R axis reduced the growth and invasion of NSCLC cells via inhibiting Wnt/β‑catenin signaling and PD‑L1 expression. The present results may provide a novel molecular target for NSCLC diagnosis and therapy.