Downregulation of PDIA3 inhibits gastric cancer cell growth through cell cycle regulation.

OBJECTIVE: Protein disulfide isomerase A3 (PDIA3) promotes the correct folding of newly synthesized glycoproteins in the endoplasmic reticulum. PDIA3 is overexpressed in most tumors, and it may become a biomarker of cancer prognosis and immunotherapy. Our study aims to detect the expression level of PDIA3 in gastric cancer (GC) and its association with GC development as wells as the underlying mechanisms. METHODS: GC cell lines with PDIA3 knockdown by siRNA, CRISPR-cas9 sgRNAs or a pharmacological inhibitor of LOC14 were prepared and used. PDIA3 knockout GC cells were established by CRISPR-cas9-PDIA3 system. The proliferation, migration, invasion and cell cycle of GC cells were analyzed by cell counting kit-8 assay, wound healing assay, transwell assay and flow cytometry, respectively. Immunodeficient nude mice was used to evaluate the role of PDIA3 in tumor formation. Quantitative PCR and western blot were used for examining gene and protein expressions. RNA sequencing was performed to see the altered gene expression. RESULTS: The expressions of PDIA3 in GC tissues and cells were increased significantly, and its expression was negatively correlated with the three-year survival rate of GC patients. Down-regulation of PDIA3 by siRNA, LOC14 or CRISPR-cas9 significantly inhibited proliferation, invasion and migration of GC cells TMK1 and AGS, with cell cycle arrested at G2/M phase. Meanwhile, decreased PDIA3 significantly inhibited growth of tumor xenograft in vivo. It was found that cyclin G1 (encoded by CCNG1 gene) expression was decreased by downregulation of PDIA3 in GC cells both in vitro and in vivo. In addition, protein levels of other cell cycle related factors including cyclin D1, CDK2, and CDK6 were also significantly decreased. Further study showed that STAT3 was associated with PDIA3-mediated cyclin G1 regulation. CONCLUSION: PDIA3 plays an oncogenic role in GC. Our findings unfolded the functional role of PDIA3 in GC development and highlighted a novel target for cancer therapeutic strategy.

Early-stage CCNG1+ HR+ HER2+ Invasive Breast Carcinoma in Older Women: Current Treatment and Future Perspectives for DeltaRex-G, a CCNG1 Inhibitor.

Women with HR+HER2+ early-stage breast cancer are disadvantaged by the lack of clinical trials focused on women ≥70 years of age. In the past years, there has been increasing controversy on the use of toxic chemotherapy as standard of care treatment for early- stage HR+ HER2+ breast carcinoma in older women. With precision medicine coming of age, molecular profiling of tumors and circulating tumor DNA has identified target oncogenes that could be used in designing an optimal treatment for this group of women. This article reviews the current treatment of early-stage triple receptor positive breast cancer, the risks of chemotherapy in older women, and CCNG1, a novel biomarker in development for the use of DeltaRex-G, a CCNG1 inhibitor. Further, future perspectives for DeltaRex-G in older women with early stage CCNG1+ HR+ HER2+ breast cancer are discussed.

Silencing of LINC00467 inhibits cell proliferation in testicular germ cell tumors cells.

A significant decrease in LINC00467 expression in testicular germ cell tumors (TGCTs) was found in our previous study in comparison to adjacent tissue. Interestingly, the expression of LINC00467 correlated with the pathological grade of the tumor in TGCT patients. The higher the expression of LINC00467 was, the worse the prognosis of the patients with TGCT was. Despite these findings, the exact role of LINC00467 in the development of TGCTs requires further investigation. LINC00467 expression was downregulated in the NCCIT and TCam-2 cell lines via small interfering RNA (siRNA) silencing. The levels of gene expression were validated using quantitative real-time polymerase chain reaction (qRT-PCR) analyses. Cell proliferation was evaluated by the MTT and Cell Counting Kit-8 (CCK8) assays, whereas flow cytometry was used to assess the effects on the cell cycle. Western blotting analysis was used to detect expression levels of protein. Additionally, RNA-sequencing and bioinformatics methods were used to investigate the mechanism of action of LINC00467 in TGCTs. The suppression of LINC00467 expression resulted in decreased cell proliferation and induced S-phase arrest. Furthermore, the suppression of LINC00467 downregulated proliferating cell nuclear antigen (PCNA), a protein related to cell cycle regulation, while it upregulated p21 expression. In other studies involving dihydrotestosterone (DHT) stimulation, it was observed that DHT could upregulate LINC00467 expression. In addition, silencing of the LINC00467 reversed the effect of testosterone on cell proliferation. The Gene Set Enrichment Analysis (GSEA) revealed that LINC00467 regulated the p53 pathway by modulating the expression of CCNG1. Our study found that LINC00467 regulates cell proliferation by inducing S-phase arrest through the cell cycle-related proteins PCNA and p21. These findings contribute to our understanding of non-coding RNAs mechanisms involved in the development of TGCTs.

PRMT6 functionally associates with PRMT5 to promote colorectal cancer progression through epigenetically repressing the expression of CDKN2B and CCNG1.

BACKGROUND: Protein arginine methyltransferase 6 (PRMT6) is a type I arginine methyltransferase that asymmetrically dimethylates histone H3 arginine 2 (H3R2me2a). However, the biological roles and underlying molecular mechanisms of PRMT6 in colorectal cancer (CRC) remain unclear. METHODS: PRMT6 expression in CRC tissue was examined using immunohistochemistry. The effect of PRMT6 on CRC cells was investigated in vitro and in vivo. Mass spectrometry, co-immunoprecipitation and GST pulldown assays were performed to identify interaction partners of PRMT6. RNA-seq, chromatin immunoprecipitation, Western blot and qRT-PCR assays were used to investigate the mechanism of PRMT6 in gene regulation. RESULTS: PRMT6 is significantly upregulated in CRC tissues and facilitates cell proliferation of CRC cells in vitro and in vivo. Through RNA-seq analysis, CDKN2B (p15INK4b) and CCNG1 were identified as new transcriptional targets of PRMT6. PRMT6-dependent H3R2me2a mark was predominantly deposited at the promoters of CDKN2B and CCNG1 in CRC cells. Furthermore, PRMT5 was firstly characterized as an interaction partner of PRMT6. Notably, H3R2me2a coincides with PRMT5-mediated H4R3me2s and H3R8me2s marks at the promoters of CDKN2B and CCNG1 genes, thus leading to transcriptional repression of these genes. CONCLUSIONS: PRMT6 functionally associates with PRMT5 to promote CRC progression through epigenetically repressing the expression of CDKN2B and CCNG1. These insights raise the possibility that combinational intervention of PRMT6 and PRMT5 may be a promising strategy for CRC therapy.

Cyclin G1 induces maladaptive proximal tubule cell dedifferentiation and renal fibrosis through CDK5 activation.

Acute kidney injury (AKI) occurs in approximately 13% of hospitalized patients and predisposes patients to chronic kidney disease (CKD) through the AKI-to-CKD transition. Studies from our laboratory and others have demonstrated that maladaptive repair of proximal tubule cells (PTCs), including induction of dedifferentiation, G2/M cell cycle arrest, senescence, and profibrotic cytokine secretion, is a key process promoting AKI-to-CKD transition, kidney fibrosis, and CKD progression. The molecular mechanisms governing maladaptive repair and the relative contribution of dedifferentiation, G2/M arrest, and senescence to CKD remain to be resolved. We identified cyclin G1 (CG1) as a factor upregulated in chronically injured and maladaptively repaired PTCs. We demonstrated that global deletion of CG1 inhibits G2/M arrest and fibrosis. Pharmacological induction of G2/M arrest in CG1-knockout mice, however, did not fully reverse the antifibrotic phenotype. Knockout of CG1 did not alter dedifferentiation and proliferation in the adaptive repair response following AKI. Instead, CG1 specifically promoted the prolonged dedifferentiation of kidney tubule epithelial cells observed in CKD. Mechanistically, CG1 promotes dedifferentiation through activation of cyclin-dependent kinase 5 (CDK5). Deletion of CDK5 in kidney tubule cells did not prevent G2/M arrest but did inhibit dedifferentiation and fibrosis. Thus, CG1 and CDK5 represent a unique pathway that regulates maladaptive, but not adaptive, dedifferentiation, suggesting they could be therapeutic targets for CKD.

miR-488-3p Protects Cardiomyocytes against Doxorubicin-Induced Cardiotoxicity by Inhibiting CyclinG1.

OBJECTIVE: To investigate the protective effects and regulatory mechanism of miR-488-3p on doxorubicin-induced cardiotoxicity. METHODS: The C57BL/6 mice and primary cardiomyocytes were used to construct doxorubicin-induced cardiomyocyte injury models in vivo and in vitro. The levels of miR-488-3p and its downstream target genes were analyzed by quantitative real-time PCR. Mouse cardiac function, cell survival, cellular injury-related proteins, and the apoptosis level of cardiomyocytes were analyzed by echocardiography, MTT analysis, Western blotting, and DNA laddering separately. RESULTS: Cardiomyocyte injury caused by a variety of stimuli can lead to the reduction of miR-488-3p level, especially when stimulated with doxorubicin. Doxorubicin led to significant decrease in cardiac function, cell autophagic flux blockage, and apoptosis in vivo and in vitro. The expression of miR-488-3p's target gene, CyclinG1, increased remarkably in the doxorubicin-treated neonatal mouse cardiomyocytes. Overexpression of miR-488-3p inhibited CyclinG1 expression, increased cardiomyocyte viability, and attenuated doxorubicin-induced cardiomyocyte autophagic flux blockage and apoptosis. CONCLUSIONS: miR-488-3p is one of the important protective miRNAs in doxorubicin-induced cardiotoxicity by inhibiting the expression of CyclinG1, which provides insight into the possible clinical application of miR-488-3p/CyclinG1 as therapeutic targets in doxorubicin-induced cardiovascular diseases.

lncRNA MSC-AS1 Aggravates Diabetic Nephropathy by Regulating the miR-325/CCNG1 Axis.

BACKGROUND: Diabetic nephropathy (DN) is the most common microvascular complication of diabetes and has become the second leading cause of end-stage renal disease in the world. This study aims to clarify the regulatory mechanism of the lncRNA MSC-AS1/miR-325/cyclin G1 (CCNG1) axis in DN. METHODS: The regulatory mechanism of lncRNA MSC-AS1/miR-325/CCNG1 was evaluated by RT-qPCR, CCK-8 assay, flow cytometry assay, RNA pull-down assay, ELISA, and western blot assay. RESULTS: Upregulation of lncRNA MSC-AS1 was detected in DN patients and HRMC cells treated with high glucose (HG). Knockdown of lncRNA MSC-AS1 reduced the proliferation, fibrosis, and inflammation of HRMC cells induced by HG. In addition, lncRNA MSC-AS1 acts as a miR-325 sponge in the DN. CCNG1 is the direct target of miR-325, which can be positively regulated by lncRNA MSC-AS1 in DN. More importantly, downregulation of miR-325 and upregulation of CCNG1 can attenuate the protective effect of lncRNA MSC-AS1 knockdown on DN. CONCLUSION: lncRNA MSC-AS1 aggravates DN by downregulating miR-325 and upregulating CCNG1.

MiR-122 radiosensitize hepatocellular carcinoma cells by suppressing cyclin G1.

PURPOSE: Emerging evidence has shown that radiotherapy is an effective treatment for hepatocellular carcinoma (HCC), Micro(mi)RNAs are involved in regulating radiosensitivity in many cancers. MiR-122 accounts for approximately 70% of all cloned miRNAs in the liver, but there are few reports about whether it is involved in regulating of radiosensitivity in HCC cells. MATERIALS AND METHODS: HCC cells (HepG2 and Huh7) overexpressing miR-122 were constructed by transfecting them with lentiviral-miR-122. Then, their proliferation ability was analyzed by the MTT, and colony formation assays and a xenograft tumor model was used to detect their radiosensitivity. The expression of cyclin G1 mRNA and protein was detected by the quantitative real-time polymerase chain reaction and western blotting, respectively. RESULTS: Overexpression of miR-122 inhibited the proliferation of, and radiosensitized HCC cells. Cyclin G1 mRNA and protein level were suppressed in HepG2 tumors overexpression miR-122. CONCLUSION: MiR-122 may be useful as a potential radiosensitizer for HCC, and its mechanism is related to the regulation of cyclin G1.

The stimulative function of long noncoding RNA CDKN2B-AS1 in osteosarcoma by targeting the microRNA-122/CCNG1 axis.

Osteosarcoma (OS), a prevalent aggressive malignancy in the bone, has limited therapeutic targets and diagnostic biomarkers. In the current investigation, RT-qPCR showed that CDKN2B-AS1 was enhanced in OS samples and cells. This research was set to examine the modulation of CDKN2B-AS1 in OS. The expression of CDKN2B-AS1 and downstream molecules was analyzed by RT-qPCR method. CCK8, EdU staining along with Transwell assays were applied to evaluate cell proliferation and invasion. Those in vitro investigations specified that silencing of CDKN2B-AS1 with shRNAs obviously impeded the proliferation and invasion of MG63 cells. To authenticate the relationships between CDKN2B-AS1 and microRNA-122-5p (miR-122-5p) or cyclin G1 (CCNG1) and miR-122-5p, we next employed luciferase reporter assay. We displayed that CDKN2B-AS1 repressed miR-122-5p to restore CCNG1 expression. All in all, our findings substantiated the indispensable function of CDKN2B-AS1 in OS progression and the possible molecular mechanism.

LncRNACASC9 promotes proliferation, metastasis, and cell cycle inovarian carcinoma cells through cyclinG1/TP53/MMP7 signaling.

Ovarian cancer (OC) brings about serious physical and psychological burden for female patients. LncRNA CASC9 has been reported to be intimately linked with the occurrence and development of several tumors. However, the biological role of lncRNA CASC9 in OC still lacks sufficient evidence. The expressions of CASC9 and miR-488-3p in OC cell lines and xenograft mice were detected by qRT-PCR assay. Cell Counting Kit-8 (CCK-8) assay was used to assess cell inhibition rate and cell proliferation in OVCAR-3 and OVCAR-3/DDP cells. Wound healing assay and transwell assay were performed to evaluate the capacity of migration and invasion, respectively. In addition, cell apoptosis was measured by TUNEL assay and cell cycle was assessed by flow cytometric analysis. Moreover, western blotting was carried out to detect the cyclinG1 (CCNG1)/TP53/MMP7 signaling and apoptosis-related proteins. Furthermore, luciferase reporter assay was performed to verify the combination of CASC9 with CCNG1 and miR-488-3p. The results of our study revealed that CASC9 expression was upregulated while miR-488-3p and CCNG1 expression was downregulated in OC cells with significant higher TP53 and MMP7 protein levels compared with normal ovarian surface epithelial cells. Additionally, luciferase reporter assay confirmed CASC9 bond to miR-488-3p/CCNG1. CASC9 silencing inhibited cell proliferation, migration, and invasion whereas promoted cell inhibition rate and apoptosis in vitro and in vivo. However, CASC9 overexpression showed the opposite effects. In summary, LncRNA CASC9 played a regulative role in ovarian carcinoma by cyclinG1/TP53/MMP7 signaling via binding to miR-488-3p in vivo and in vitro.

The dietary carcinogen PhIP activates p53-dependent DNA damage response in the colon of CYP1A-humanized mice.

Species differences in the metabolism of xenobiotics by cytochrome P450 are critical in evaluating the use of experimental animals in studying toxic compounds relevant to human diseases. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which is produced by high-temperature cooking of fish and meat, is activated to become a carcinogen by cytochrome P4501A2 (CYP1A2) through N2 -hydroxylation in humans, but is detoxified by Cyp1a2 through 4'-hydroxylation in mice. CYP1A-humanized (hCYP1A) mice, in which mouse Cyp1a is replaced with human CYP1A, show constitutive human xenobiotic metabolism by hCYP1A, thereby serving as a suitable model for studying PhIP. Previous studies have demonstrated that oral administration of PhIP induces colon tumors in hCYP1A mice; however, these studies used a super-high dose, raising concerns regarding the relevance of the mechanism to human cancer. Herein, we systematically investigated PhIP-induced colon carcinogenesis in hCYP1A mice treated with lower doses. We found that a dose 2000 times lower than that used previously, which is comparable to human daily intake levels, could induce colon tumors, albeit at a lower incidence rate. We further investigated the transcriptome changes in the colon of hCYP1A mice treated with PhIP and identified that PhIP treatment increased the expression of Bax, Btg2, Ccng1, Cdkn1a, and Trp53inp1 and decreased the expression of Igf1 and Ccnd1. Since these genes are key components of the p53-dependent DNA damage response, the altered expression patterns indicated PhIP-induced DNA damage in hCYP1A mice. Together, these results prove that hCYP1A mice are suitable for studying PhIP-induced carcinogenesis and show that PhIP is an important colorectal cancer carcinogen in human diet.

Long non­coding RNA OIP5­AS1 facilitates the progression of ovarian cancer via the miR­128­3p/CCNG1 axis.

Long non­coding RNA (LncRNA) o­phthalaldehyde-interacting protein 5 antisense transcript 1 (OIP5­AS1) serves major roles in the progression of various types of cancer. The present study investigated its biological function in ovarian cancer (OC) and its mechanisms. The levels of OIP5­AS1, microRNA­128­3p (miR­128­3p) and cyclin G1 (CCNG1) were examined by reverse transcription­quantitative PCR. Cell viability, apoptosis, migration and invasion were detected to analyze cellular progression. Glycolytic metabolism was assessed by detecting the levels of glucose consumption and lactate production. CCNG1 and hexokinase 2 protein levels were measured by western blotting. Dual­luciferase reporter assay, RNA immunoprecipitation and RNA pull­down assays were performed to affirm the interaction between two molecules. OIP5­AS1 was found to be upregulated in OC tissues and cells. Knockdown of OIP5­AS1 suppressed cell viability, migration, invasion and glycolysis while promoting apoptosis in OC cells. OIP5­AS1 interacted with miR­128­3p and functioned as an oncogene by sequestering miR­128­3p. In addition, CCNG1 was a target gene for miR­128­3p and miR­128­3p regulated the CCNG1­induced effects on OC cells by downregulating CCNG1. OIP5­AS1 upregulated the expression of CCNG1 via targeting miR­128­3p. OIP5­AS1 knockdown also inhibited tumor growth of OC in vivo by modulating the expression of miR­128­3p and CCNG1. Collectively, these data illustrated that the oncogenic role of OIP5­AS1 in OC was associated with the miR­128­3p/CCNG1 axis at least in part. OIP5­AS1 might be a probable diagnostic and therapeutic biomarker for the treatment of OC patients.

Silencing the Expression of Cyclin G1 Enhances the Radiosensitivity of Hepatocellular Carcinoma In Vitro and In Vivo by Inducing Apoptosis.

Radiotherapy plays an important role in the treatment of hepatocellular carcinoma (HCC). Cyclin G1 is a novel member of the cyclin family, and it is abnormally expressed in HCC. In this study we investigated the role of cyclin G1 in the radiotherapy of HCC cells. The expression of cyclin G1 was silenced by transfection of cyclin G1-siRNA into HepG2 cells and Huh7 cells, and the expression of cyclin G1 mRNA and protein was measured by qRT-PCR and Western blot analysis. The proliferation was analyzed using MTT assay, and the radiosensitivity of HCC cells was detected using colony formation assay and a xenograft tumor model. The expression of apoptosis-related proteins (Bcl-2 and Bax) was detected by Western blot analysis, and caspase-3 was detected using fluorimetry. The expression of cyclin G1 mRNA and protein in HepG2/Huh7-cyclin G1-siRNA cells was found to be significantly decreased compared to that in HepG2/Huh7 cells. Silencing the expression of cyclin G1 inhibited the proliferation of HCC cells and enhanced radiosensitivity in HCC cells in vitro and in vivo. Knockdown of cyclin G1 expression significantly decreased Bcl-2 expression, and increased Bax expression and caspase-3 activity in HCC cells. Silencing of cyclin G1 expression enhances the radiosensitivity of HCC cells in vitro and in vivo. The mechanism for this may be related to the regulation of apoptosis-related proteins.

Cyclin G2 upregulation impairs migration, invasion, and network formation through RNF123/Dvl2/JNK signaling in the trophoblast cell line HTR8/SVneo, a possible role in preeclampsia.

Disruption of extravillous trophoblast (EVT) migration and invasion is considered to be responsible for pathological placentation in preeclampsia (PE). Cyclin G2 (CCNG2) is an atypical cyclin that inhibits cell cycle progression. However, its biological function and underlying molecular mechanism in PE are poorly understood. In this study, clinical data demonstrated that CCNG2 was significantly upregulated in PE placenta and associated with invasive EVT dysfunction. Additionally, Ccng2 knockout led to an attenuation of PE-like symptoms in the PE mouse model produced via treatment with NG-nitro-L-arginine methyl ester (L-NAME). In vitro, CCNG2 inhibited the migration, invasion, and endothelial-like network formation of human trophoblast cell line HTR8/SVneo. Mechanically, CCNG2 suppressed JNK-dependent Wnt/PCP signaling and its downstream indicators including epithelial-to-mesenchymal transition (EMT) markers and matrix metalloproteinases (MMPs) via promoting the polyubiquitination degradation of dishevelled 2 (Dvl2) protein in HTR8/SVneo cells. We also discovered that the E3 ligase Ring finger protein 123 (RNF123), as a novel CCNG2 target among HTR8/SVneo cells, interacted with Dvl2 and participated in CCNG2-induced polyubiquitination degradation of Dvl2. Moreover, we verified that the treatment of HTR8/SVneo cells with RNF123-specific siRNA improved polyubiquitination-induced degradation of Dvl2 and the activity of Wnt/PCP-JNK signaling mediated by CCNG2. Taken together, our results reveal that the CCNG2/RNF123/Dvl2/JNK axis may be involved in the pathogenesis and progression of PE through trophoblastic cell function modulation, thus probably providing us with new therapeutic strategies for PE treatment.

Loss of Sbds in zebrafish leads to neutropenia and pancreas and liver atrophy.

Shwachman-Diamond syndrome (SDS) is characterized by exocrine pancreatic insufficiency, neutropenia, and skeletal abnormalities. Biallelic mutations in SBDS, which encodes a ribosome maturation factor, are found in 90% of SDS cases. Sbds-/- mice are embryonic lethal. Using CRISPR/Cas9 editing, we created sbds-deficient zebrafish strains. Sbds protein levels progressively decreased and became undetectable at 10 days postfertilization (dpf). Polysome analysis revealed decreased 80S ribosomes. Homozygous mutant fish developed normally until 15 dpf. Mutant fish subsequently had stunted growth and showed signs of atrophy in pancreas, liver, and intestine. In addition, neutropenia occurred by 5 dpf. Upregulation of tp53 mRNA did not occur until 10 dpf, and inhibition of proliferation correlated with death by 21 dpf. Transcriptome analysis showed tp53 activation through upregulation of genes involved in cell cycle arrest, cdkn1a and ccng1, and apoptosis, puma and mdm2. However, elimination of Tp53 function did not prevent lethality. Because of growth retardation and atrophy of intestinal epithelia, we studied the effects of starvation on WT fish. Starved WT fish showed intestinal atrophy, zymogen granule loss, and tp53 upregulation - similar to the mutant phenotype. In addition, there was reduction in neutral lipid storage and ribosomal protein amount, similar to the mutant phenotype. Thus, loss of Sbds in zebrafish phenocopies much of the human disease and is associated with growth arrest and tissue atrophy, particularly of the gastrointestinal system, at the larval stage. A variety of stress responses, some associated with Tp53, contribute to pathophysiology of SDS.

MicroRNA-488 inhibits ovarian cancer cell metastasis through regulating CCNG1 and p53 expression.

OBJECTIVE: The roles of microRNAs (miRNAs) have been widely exploited in cancer. MiRNAs have become a potential breakthrough in cancer diagnosis and treatment. Here, the regulatory mechanism of microRNA-488 (miR-488) was investigated in ovarian cancer (OC). PATIENTS AND METHODS: The expression levels of miR-488 and CCNG1 (Cyclin G1) were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot assays. Transwell assay and epithelial-mesenchymal transition (EMT) markers were used to clarify the effect of miR-488 on cell metastasis. The dual-luciferase reporter assay was used to verify the relation between miR-488 and CCNG1. RESULTS: The expression of miR-488 was reduced in OC, which was associated with poor clinical outcomes and prognosis in OC patients. MiR-488 inhibited cell metastasis in OC by blocking EMT and promoting tumor suppressor p53 expression. In addition, CCNG1 was confirmed as a direct target of miR-488. Upregulation of CCNG1 impaired the inhibitory effect of miR-488 in OC. CONCLUSIONS: MiR-488 serves as a tumor inhibitor in OC by suppressing cell metastasis, indicating that miR-488 has a great potential in the diagnosis and treatment of OC.

Locally acting transcription factors regulate p53-dependent cis-regulatory element activity.

The master tumor suppressor p53 controls transcription of a wide-ranging gene network involved in apoptosis, cell cycle arrest, DNA damage repair, and senescence. Recent studies revealed pervasive binding of p53 to cis-regulatory elements (CREs), which are non-coding segments of DNA that spatially and temporally control transcription through the combinatorial binding of local transcription factors. Although the role of p53 as a strong trans-activator of gene expression is well known, the co-regulatory factors and local sequences acting at p53-bound CREs are comparatively understudied. We designed and executed a massively parallel reporter assay (MPRA) to investigate the effect of transcription factor binding motifs and local sequence context on p53-bound CRE activity. Our data indicate that p53-bound CREs are both positively and negatively affected by alterations in local sequence context and changes to co-regulatory TF motifs. Our data suggest p53 has the flexibility to cooperate with a variety of transcription factors in order to regulate CRE activity. By utilizing different sets of co-factors across CREs, we hypothesize that global p53 activity is guarded against loss of any one regulatory partner, allowing for dynamic and redundant control of p53-mediated transcription.

Silencing CCNG1 protects MPC-5 cells from high glucose-induced proliferation-inhibition and apoptosis-promotion via MDM2/p53 signaling pathway.

PURPOSE: Diabetic nephropathy (DN) is one of the most serious complications of diabetes mellitus and one of the most important causes of end-stage renal disease, but its pathogenesis has not been elucidated so far, and there is no effective treatment. METHODS: DN models of rats and MPC-5 cells were established with streptozotocin (STZ) and high glucose (HG) in vivo and in vitro, respectively. Cell markers desmin and nephrin in foot kidney tissue were detected by Western blot. CCNG1 level in vitro was analyzed by Western blot and immunohistochemistry. CCK-8 assay and flow cytometry were conducted to analyze the effect of CCNG1 on HG-treated MPC-5 cells. Apoptosis-related proteins (Bcl-2, Bax and p53), CCNG1, and MDM2 were determined by RT-qPCR and Western blot. RESULTS: The level of nephrin was decreased, while desmin was increased in STZ-induced DN rats and CCNG1 level was also enhanced by STZ. In vitro experiments indicated that MPC-5 cell viability was inhibited and apoptosis was induced by HG and we also found that CCNG1 expression was up-regulated by HG and negatively correlated with MDM2 level. The effects of HG on MPC-5 cell viability, apoptosis, and cell cycle were reversed by silencing CCNG1, but further deteriorated by overexpression of CCNG1. Furthermore, overexpression of MDM2 inhibited HG-induced MPC-5 cell injury and CCNG1 expression. CONCLUSIONS: These findings revealed that down-regulation of CCNG1 has protection effects in DN that is mechanistically linked to MDM2-p53 pathways.

Upregulation of microRNA-23b-3p induced by farnesoid X receptor regulates the proliferation and apoptosis of osteosarcoma cells.

BACKGROUND: The downstream targets of farnesoid X receptor (FXR) such as miRNAs have a potent effect on the progression of many types of cancer. We aim to study the effects of FXR on osteosarcoma (OS) development and the potential role of microRNA-23b-3p. METHODS: The expressions of FXR and miR-23b-3p in normal osteoblasts and five osteosarcoma cell lines were measured. Their correlations were analyzed by Pearson's test and verified by the introduction of FXR agonist, GW4064. TargetScan predicted that cyclin G1 (CCNG1) was a target for miR-23b-3p. The transfection of FXR siRNA was performed to confirm the correlation between FXR and miR-23b-3p. We further transfected miR-23b-3p inhibitor into MG-63 cells, and the transfected cells were treated with 5 µM GW4064 for 48 h. Quantitative PCR (qPCR) and Western blot were performed for expression analysis. Cell proliferation, cell apoptosis rate, and cell cycle distribution were assessed by clone formation assay and flow cytometry. RESULTS: Scatter plot showed a positive correlation between FXR and miR-23b-3p (Pearson's coefficient test R2 = 1.00, P = 0.0028). As CCNG1 is a target for miR-23b-3p, the treatment of GW4064 induced the downregulation of CCNG1 through upregulating miR-23b-3p. The inhibition of miR-23b-3p obviously promoted cell viability, proliferation, and cell cycle progression but reduced apoptosis rate of MG-63 cells; however, the treatment of GW4064 could partially reverse the effects of the inhibition of miR-23b-3p on OS cells. CONCLUSIONS: Upregulated FXR by GW4064 can obviously suppress OS cell development, and the suppressive effects may rely on miR-23b-3p/CCNG1 pathway.