Toll-like receptors TLR2 and TLR4 block the replication of pancreatic ß cells in diet-induced obesity.

Consumption of a high-energy Western diet triggers mild adaptive ß cell proliferation to compensate for peripheral insulin resistance; however, the underlying molecular mechanism remains unclear. In the present study we show that the toll-like receptors TLR2 and TLR4 inhibited the diet-induced replication of ß cells in mice and humans. The combined, but not the individual, loss of TLR2 and TLR4 increased the replication of ß cells, but not that of α cells, leading to enlarged ß cell area and hyperinsulinemia in diet-induced obesity. Loss of TLR2 and TLR4 increased the nuclear abundance of the cell cycle regulators cyclin D2 and Cdk4 in a manner dependent on the signaling mediator Erk. These data reveal a regulatory mechanism controlling the proliferation of ß cells in diet-induced obesity and suggest that selective targeting of the TLR2/TLR4 pathways may reverse ß cell failure in patients with diabetes.

Generation of adult hippocampal neural stem cells occurs in the early postnatal dentate gyrus and depends on cyclin D2.

Lifelong hippocampal neurogenesis is maintained by a pool of multipotent adult neural stem cells (aNSCs) residing in the subgranular zone of the dentate gyrus (DG). The mechanisms guiding transition of NSCs from the developmental to the adult state remain unclear. We show here, by using nestin-based reporter mice deficient for cyclin D2, that the aNSC pool is established through cyclin D2-dependent proliferation during the first two weeks of life. The absence of cyclin D2 does not affect normal development of the dentate gyrus until birth but prevents postnatal formation of radial glia-like aNSCs. Furthermore, retroviral fate mapping reveals that aNSCs are born on-site from precursors located in the dentate gyrus shortly after birth. Taken together, our data identify the critical time window and the spatial location of the precursor divisions that generate the persistent population of aNSCs and demonstrate the central role of cyclin D2 in this process.

CRL4AMBRA1 is a master regulator of D-type cyclins.

D-type cyclins are central regulators of the cell division cycle and are among the most frequently deregulated therapeutic targets in human cancer1, but the mechanisms that regulate their turnover are still being debated2,3. Here, by combining biochemical and genetics studies in somatic cells, we identify CRL4AMBRA1 (also known as CRL4DCAF3) as the ubiquitin ligase that targets all three D-type cyclins for degradation. During development, loss of Ambra1 induces the accumulation of D-type cyclins and retinoblastoma (RB) hyperphosphorylation and hyperproliferation, and results in defects of the nervous system that are reduced by treating pregnant mice with the FDA-approved CDK4 and CDK6 (CDK4/6) inhibitor abemaciclib. Moreover, AMBRA1 acts as a tumour suppressor in mouse models and low AMBRA1 mRNA levels are predictive of poor survival in cancer patients. Cancer hotspot mutations in D-type cyclins abrogate their binding to AMBRA1 and induce their stabilization. Finally, a whole-genome, CRISPR-Cas9 screen identified AMBRA1 as a regulator of the response to CDK4/6 inhibition. Loss of AMBRA1 reduces sensitivity to CDK4/6 inhibitors by promoting the formation of complexes of D-type cyclins with CDK2. Collectively, our results reveal the molecular mechanism that controls the stability of D-type cyclins during cell-cycle progression, in development and in human cancer, and implicate AMBRA1 as a critical regulator of the RB pathway.

Methylation of KSHV vCyclin by PRMT5 contributes to cell cycle progression and cell proliferation.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a double-stranded DNA virus that encodes numerous cellular homologs, including cyclin D, G protein-coupled protein, interleukin-6, and macrophage inflammatory proteins 1 and 2. KSHV vCyclin encoded by ORF72, is the homolog of cellular cyclinD2. KSHV vCyclin can regulate virus replication and cell proliferation by constitutively activating cellular cyclin-dependent kinase 6 (CDK6). However, the regulatory mechanism of KSHV vCyclin has not been fully elucidated. In the present study, we identified a host protein named protein arginine methyltransferase 5 (PRMT5) that interacts with KSHV vCyclin. We further demonstrated that PRMT5 is upregulated by latency-associated nuclear antigen (LANA) through transcriptional activation. Remarkably, knockdown or pharmaceutical inhibition (using EPZ015666) of PRMT5 inhibited the cell cycle progression and cell proliferation of KSHV latently infected tumor cells. Mechanistically, PRMT5 methylates vCyclin symmetrically at arginine 128 and stabilizes vCyclin in a methyltransferase activity-dependent manner. We also show that the methylation of vCyclin by PRMT5 positively regulates the phosphorylate retinoblastoma protein (pRB) pathway. Taken together, our findings reveal an important regulatory effect of PRMT5 on vCyclin that facilitates cell cycle progression and proliferation, which provides a potential therapeutic target for KSHV-associated malignancies.

ErbB3 is required for hyperaminoacidemia-induced pancreatic α cell hyperplasia.

Blood amino acid levels are maintained in a narrow physiological range. The pancreatic α cells have emerged as the primary aminoacidemia regulator through glucagon secretion to promote hepatic amino acid catabolism. Interruption of glucagon signaling disrupts the liver-α cells axis leading to hyperaminoacidemia, which triggers a compensatory rise in glucagon secretion and α cell hyperplasia. The mechanisms of hyperaminoacidemia-induced α cell hyperplasia remain incompletely understood. Using a mouse α cell line and in vivo studies in zebrafish and mice, we found that hyperaminoacidemia-induced α cell hyperplasia requires ErbB3 signaling. In addition to mechanistic target of rapamycin complex 1, another ErbB3 downstream effector signal transducer and activator of transcription 3 also plays a role in α cell hyperplasia. Mechanistically, ErbB3 may partner with ErbB2 to stimulate cyclin D2 and suppress p27 via mechanistic target of rapamycin complex 1 and signal transducer and activator of transcription 3. Our study identifies ErbB3 as a new regulator for hyperaminoacidemia-induced α cell proliferation and a critical component of the liver-α cells axis that regulates aminoacidemia.

CCND2 Modified mRNA Activates Cell Cycle of Cardiomyocytes in Hearts With Myocardial Infarction in Mice and Pigs.

BACKGROUND: Experiments in mammalian models of cardiac injury suggest that the cardiomyocyte-specific overexpression of CCND2 (cyclin D2, in humans) improves recovery from myocardial infarction (MI). The primary objective of this investigation was to demonstrate that our specific modified mRNA translation system (SMRTs) can induce CCND2 expression in cardiomyocytes and replicate the benefits observed in other studies of cardiomyocyte-specific CCND2 overexpression for myocardial repair. METHODS: The CCND2-cardiomyocyte-specific modified mRNA translation system (cardiomyocyte SMRTs) consists of 2 modRNA constructs: one codes for CCND2 and contains a binding site for L7Ae, and the other codes for L7Ae and contains recognition elements for the cardiomyocyte-specific microRNAs miR-1 and miR-208. Thus, L7Ae suppresses CCND2 translation in noncardiomyocytes but is itself suppressed by endogenous miR-1 and -208 in cardiomyocytes, thereby facilitating cardiomyocyte-specific CCND2 expression. Experiments were conducted in both mouse and pig models of MI, and control assessments were performed in animals treated with an SMRTs coding for the cardiomyocyte-specific expression of luciferase or green fluorescent protein (GFP), in animals treated with L7Ae modRNA alone or with the delivery vehicle, and in Sham-operated animals. RESULTS: CCND2 was abundantly expressed in cultured, postmitotic cardiomyocytes 2 days after transfection with the CCND2-cardiomyocyte SMRTs, and the increase was accompanied by the upregulation of markers for cell-cycle activation and proliferation (eg, Ki67 and Aurora B kinase). When the GFP-cardiomyocyte SMRTs were intramyocardially injected into infarcted mouse hearts, the GFP signal was observed in cardiomyocytes but no other cell type. In both MI models, cardiomyocyte proliferation (on day 7 and day 3 after treatment administration in mice and pigs, respectively) was significantly greater, left-ventricular ejection fractions (days 7 and 28 in mice, days 10 and 28 in pigs) were significantly higher, and infarcts (day 28 in both species) were significantly smaller in animals treated with the CCND2-cardiomyocyte SMRTs than in any other group that underwent MI induction. CONCLUSIONS: Intramyocardial injections of the CCND2-cardiomyocyte SMRTs promoted cardiomyocyte proliferation, reduced infarct size, and improved cardiac performance in small and large mammalian hearts with MI.

Quantification of cyclin D1 and D2 proteins in multiple myeloma identifies different expression patterns from those revealed by gene expression profiling.

Upregulation of a cyclin D gene determined by expression microarrays is an almost universal event in multiple myeloma (MM), but this finding has not been properly confirmed at the protein level. For this reason, we carried out a quantitative analysis of cyclin D proteins using a capillary electrophoresis nanoimmunoassay in newly diagnosed MM patients. Exclusive expression of cyclin D1 and D2 proteins was detected in 54 of 165 (33%) and 30 of 165 (18%) of the MM patients, respectively. Of note, cyclin D1 or D2 proteins were undetectable in 41% of the samples. High levels of cyclin D1 protein were strongly associated with the presence of t(11;14) or 11q gains. Cyclin D2 protein was detected in all the cases bearing t(14;16), but in only 24% of patients with t(4;14). The presence of cyclin D2 was associated with shorter overall survival (hazard ratio =2.14; P=0.017), although patients expressing cyclin D2 protein, but without 1q gains, had a favorable prognosis. In conclusion, although one of the cyclins D is overexpressed at the mRNA level in almost all MM patients, in approximately half of the patients this does not translate into detectable protein. This suggests that cyclins D could not play an oncogenic role in a proportion of patients with MM (clinicaltrials gov. identifier: NCT01916252).

Widespread shortening of 3'UTRs by alternative cleavage and polyadenylation activates oncogenes in cancer cells.

In cancer cells, genetic alterations can activate proto-oncogenes, thereby contributing to tumorigenesis. However, the protein products of oncogenes are sometimes overexpressed without alteration of the proto-oncogene. Helping to explain this phenomenon, we found that when compared to similarly proliferating nontransformed cell lines, cancer cell lines often expressed substantial amounts of mRNA isoforms with shorter 3' untranslated regions (UTRs). These shorter isoforms usually resulted from alternative cleavage and polyadenylation (APA). The APA had functional consequences, with the shorter mRNA isoforms exhibiting increased stability and typically producing ten-fold more protein, in part through the loss of microRNA-mediated repression. Moreover, expression of the shorter mRNA isoform of the proto-oncogene IGF2BP1/IMP-1 led to far more oncogenic transformation than did expression of the full-length, annotated mRNA. The high incidence of APA in cancer cells, with consequent loss of 3'UTR repressive elements, suggests a pervasive role for APA in oncogene activation without genetic alteration.

Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model.

Therapeutic strategies based on modulation of microRNA (miRNA) activity hold great promise due to the ability of these small RNAs to potently influence cellular behavior. In this study, we investigated the efficacy of a miRNA replacement therapy for liver cancer. We demonstrate that hepatocellular carcinoma (HCC) cells exhibit reduced expression of miR-26a, a miRNA that is normally expressed at high levels in diverse tissues. Expression of this miRNA in liver cancer cells in vitro induces cell-cycle arrest associated with direct targeting of cyclins D2 and E2. Systemic administration of this miRNA in a mouse model of HCC using adeno-associated virus (AAV) results in inhibition of cancer cell proliferation, induction of tumor-specific apoptosis, and dramatic protection from disease progression without toxicity. These findings suggest that delivery of miRNAs that are highly expressed and therefore tolerated in normal tissues but lost in disease cells may provide a general strategy for miRNA replacement therapies.

Up-regulation of lncRNA WT1-AS ameliorates Aß-stimulated neuronal injury through modulation of miR-186-5p/CCND2 axis in Alzheimer's disease.

As a common neurodegenerative disorder, Alzheimer's disease (AD) seriously threatens human life. Long non-coding RNAs (lncRNAs) exhibit essential functions in AD development. Nevertheless, the detailed effects and possible mechanisms of lncRNA Wilms tumor 1 Antisense RNA (WT1-AS) in AD are largely unknown. In our studies, a total of 30 serum samples from AD patients were collected, and WT1-AS expressions were detected through qRT-PCR analysis. Additionally, an in vitro AD model was constructed by treating Aß1-42 in human neuroblastoma cells. Functional assays were implemented to assess the impacts of WT1-AS on Aß1-42-stimulated human neuroblastoma cell proliferation together with apoptosis. Moreover, relationship of WT1-AS, microRNA (miR)-186-5p as well as cyclin D2 (CCND2) could be predicted through bioinformatics tools as well as proved via dual-luciferase reporter experiments. Our results showed that WT1-AS together with CCND2 were low-expressed, while miR-186-5p presented high expression in AD serum samples together with Aß1-42-stimulated human neuroblastoma cells. WT1-AS over-expression or miR-186-5p depletion notably promoted the proliferation, reduced the apoptosis, and decreased the p-Tau protein expressions of human neuroblastoma cells induced with Aß1-42. Moreover, miR-186-5p combined with WT1-AS, and CCND2 was modulated by miR-186-5p. Furthermore, CCND2 elevation partially offsets the impacts of miR-186-5p elevation on Aß1-42-stimulated cell proliferation as well as apoptosis mediated with WT1-AS up-regulation. Our results indicated that up-regulation of lncRNA WT1-AS ameliorated Aß-stimulated neuronal damage through modulating miR-186-5p/CCND2 axis, offering a novel direction for AD therapy.

Doxycycline-Mediated Control of Cyclin D2 Overexpression in Human-Induced Pluripotent Stem Cells.

Previous studies have demonstrated that when the cyclin D2 (CCND2), a cell-cycle regulatory protein, is overexpressed in human-induced pluripotent stem cells (hiPSCs), cardiomyocytes (CMs) differentiated from these CCND2-overexpressing hiPSCs can proliferate after transplantation into infarcted hearts, which significantly improves the cells' potency for myocardial regeneration. However, persistent CM proliferation could lead to tumor growth or the development of arrhythmogenic complications; thus, the goal of the current study was to generate a line of hiPSCs in which CCND2 overexpression could be tightly controlled. First, we transfected hiPSCs with vectors coding for a doxycycline-inducible Tet-On transactivator and S. pyogenes dCas9 fused to the VPR activation domain; then, the same hiPSCs were engineered to express guide RNAs targeting the CCND2 promotor. Thus, treatment with doxycycline (dox) activated dCas9-VPR expression, and the guide RNAs directed dCas9-VPR to the CCND2 promoter, which activated CCND2 expression. Subsequent experiments confirmed that CCND2 expression was dox-dependent in this newly engineered line of hiPSCs (doxCCND2-hiPSCs): CCND2 protein was abundantly expressed after 48 h of treatment with dox and declined to near baseline level ~96 h after dox treatment was discontinued.

Role and Regulatory Mechanism of circRNA_14820 in the Proliferation and Differentiation of Goat Skeletal Muscle Satellite Cells.

Skeletal muscle satellite cells (SMSCs), a type of myogenic stem cell, play a pivotal role in postnatal muscle regeneration and repair in animals. Circular RNAs (circRNAs) are a distinct class of non-coding RNA molecules capable of regulating muscle development by modulating gene expression, acting as microRNAs, or serving as protein decoys. In this study, we identified circ_14820, an exonic transcript derived from adenosine triphosphatase family protein 2 (ATAD2), through initial RNA-Seq analysis. Importantly, overexpression of circ_14820 markedly enhanced the proliferation of goat SMSCs while concomitantly suppressing their differentiation. Moreover, circ_14820 exhibited predominant localization in the cytoplasm of SMSCs. Subsequent small RNA and mRNA sequencing of circ_14820-overexpressing SMSCs systematically elucidated the molecular regulatory mechanisms associated with circ_14820. Our preliminary findings suggest that the circ_14820-miR-206-CCND2 regulatory axis may govern the development of goat SMSCs. These discoveries contribute to a deeper understanding of circRNA-mediated mechanisms in regulating skeletal muscle development, thereby advancing our knowledge of muscle biology.

Loss of individualized behavioral trajectories in adult neurogenesis-deficient cyclin D2 knockout mice.

There is still limited mechanistic insight into how the interaction of individuals with their environment results in the emergence of individuality in behavior and brain structure. Nevertheless, the idea that personal activity shapes the brain is implicit in strategies for healthy cognitive aging as well as in the idea that individuality is reflected in the brain's connectome. We have shown that even isogenic mice kept in a shared enriched environment (ENR) developed divergent and stable social and exploratory trajectories. As these trajectories-measured as roaming entropy (RE)-positively correlated with adult hippocampal neurogenesis, we hypothesized that a feedback between behavioral activity and adult hippocampal neurogenesis might be a causal factor in brain individualization. We used cyclin D2 knockout mice with constitutively extremely low levels of adult hippocampal neurogenesis and their wild-type littermates. We housed them for 3 months in a novel ENR paradigm, consisting of 70 connected cages equipped with radio frequency identification antennae for longitudinal tracking. Cognitive performance was evaluated in the Morris Water Maze task (MWM). With immunohistochemistry we confirmed that adult neurogenesis correlated with RE in both genotypes and that D2 knockout mice had the expected impaired performance in the reversal phase of the MWM. But whereas the wild-type animals developed stable exploratory trajectories with increasing variance, correlating with adult neurogenesis, this individualizing phenotype was absent in D2 knockout mice. Here the behaviors started out more random and revealed less habituation and low variance. Together, these findings suggest that adult neurogenesis contributes to experience-dependent brain individualization.

Chronic Exposure to Palmitic Acid Down-Regulates AKT in Beta-Cells through Activation of mTOR.

High circulating lipids occurring in obese individuals and insulin-resistant patients are considered a contributing factor to type 2 diabetes. Exposure to high lipid concentration is proposed to both protect and damage beta-cells under different circumstances. Here, by feeding mice a high-fat diet (HFD) for 2 weeks to up to 14 months, the study showed that HFD initially causes the beta-cells to expand in population, whereas long-term exposure to HFD is associated with failure of beta-cells and the inability of animals to respond to glucose challenge. To prevent the failure of beta-cells and the development of type 2 diabetes, the molecular mechanisms that underlie this biphasic response of beta-cells to lipid exposure were explored. Using palmitic acid (PA) in cultured beta-cells and islets, the study demonstrated that chronic exposure to lipids leads to reduced viability and inhibition of cell cycle progression concurrent with down-regulation of a pro-growth/survival kinase AKT, independent of glucose. This AKT down-regulation by PA is correlated with the induction of mTOR/S6K activity. Inhibiting mTOR activity with rapamycin induced Raptor and restored AKT activity, allowing beta-cells to gain proliferation capacity that was lost after HFD exposure. In summary, a novel mechanism in which lipid exposure may cause the dipole effects on beta-cell growth was elucidated, where mTOR acts as a lipid sensor. These mechanisms can be novel targets for future therapeutic developments.

Paternal sleep deprivation induces metabolic perturbations in male offspring via altered LRP5 DNA methylation of pancreatic islets.

Diabetes and metabolic perturbation are global health challenges. Sleep insufficiency may trigger metabolic dysregulation leading to diabetes. However, the intergenerational transmission of this environmental information is not clearly understood. The research objective was to determine the possible effect of paternal sleep deprivation on the metabolic phenotype of the offspring and to investigate the underlying mechanism of epigenetic inheritance. Male offspring of sleep-deprived fathers exhibit glucose intolerance, insulin resistance, and impaired insulin secretion. In these SD-F1 offspring, a reduction in beta cell mass and proliferation of beta cells were observed. Mechanistically, in pancreatic islets of SD-F1 offspring, we identified alterations in DNA methylation at the promoter region of the LRP5 (LDL receptor related protein 5) gene, a coreceptor of Wnt signaling, resulting in downregulation of downstream effectors cyclin D1, cyclin D2, and Ctnnb1. Restoration of Lrp5 in the pancreas of SD-F1 male mice could improve impaired glucose tolerance and expression of cyclin D1, cyclin D2, and Ctnnb1. This study might significantly contribute to our understanding of the effects of sleeplessness on health and metabolic disease risk from the perspective of the heritable epigenome.

MiR-188-5p inhibits cell proliferation and migration in ovarian cancer via competing for CCND2 with ELAVL1.

MicroRNAs (miRNAs) were reportedly demonstrated to participate in ovarian cancer (OC) progression. Here, we inquired into the role of miR-188-5punderneath OC cell proliferation and migration. In this respect, our work examined the miR-188-5p expression and demonstrated its expression level in OC by qRT-PCR analysis. Enforced miR-188-5p expression resulted in a serious downfall of cell growth and mobility, and acceleration apoptosis in OC cells. Furthermore, we identified CCND2 as a target gene of miR-188-5p. RIP assay and luciferase reporter assay verified the interaction of miR-188-5p and CCND2 and exhibited that miR-188-5p greatly hindered the expression of CCND2. Besides, HuR stabilized CCND2 mRNA and counteracted the miR-188-5p suppressive effect on CCND2 mRNA. Functionally, rescue experiments also showed that miR-188-5p-mediated suppression on OC cell proliferation and migration was reverted by CCND2 or HuR overexpression. Our study found miR-188-5p was a tumor suppressor in OC via competing for CCND2 with ELAVL1, contributing to coming up with novel clues for OC therapies.

Reduced expression of microRNA-432-5p by DNA methyltransferase 3B leads to development of colorectal cancer through upregulation of CCND2.

BACKGROUND: The tumor suppressive function of microRNA-432-5p (miR-432-5p) has been reported in several human malignances. This study aimed to probe the expression profile and role of miR-432-5p in colorectal cancer (CRC) and the molecular mechanism. METHODS: Differentially expressed miRNAs between CRC and healthy samples were screened using a miRNA expression dataset GSE136020. The related molecules were identified by integrated bioinformatic analyses. A murine model of primary CRC was established and xenograft tumors were induced in mice. Altered expression of DNMT3B, miR-432-5p and cyclin D2 (CCND2) were introduced in CRC cells to determine their roles in the development of CRC. RESULTS: miR-432-5p was downregulated in CRC according to the GSE136020 dataset. CCND2 mRNA was confirmed as a target of miR-432-5p. miR-432-5p was downregulated, whereas CCND2 was abundantly expressed in CRC tissues and cells. DNA methyltransferase 3B (DNMT3B) induced DNA methylation at the CpG island of miR-432-5p to inhibit its expression. miR-432-5p mimic significantly suppressed tumorigenesis of primary CRC in mice. Downregulation of DNMT3B weakened viability, invasiveness, blocked the cell cycle progression of CRC cells in vitro, and inhibited xenograft tumor growth and metastasis in nude mice. However, additional downregulation of miR-432-5p or upregulation of CCND2 restored the malignant behaviors of CRC cells. CONCLUSION: This study showed that DNMT3B induced DNA methylation and downregulation of miR-432-5p to promote development of CRC by upregulating CCND2.

CCND2 and miR-206 as potential biomarkers in the clinical diagnosis of thyroid carcinoma by fine-needle aspiration cytology.

BACKGROUND: To investigate the relationship between cyclin D2 (CCND2) and miR-206 expression in fine-needle aspiration cytology of thyroid carcinoma. METHODS: A total of 65 patients with thyroid carcinoma were selected as the subjects and 65 patients with benign thyroid nodules were in control group. The fine-needle aspiration cytology of thyroid nodules was performed. CCND2 and miR-206 levels were detected by PCR. RESULTS: Compared with the patients with benign thyroid nodules, the expression level of miR-206 in fine-needle aspiration cytology of thyroid cancer patients decreased significantly and the expression level of CCND2 increased significantly. CCND2 and miR-206 expression was negatively correlated in thyroid cancer tissues. Area under curve (AUC) of miR-206 level in the diagnosis of thyroid cancer was 0.889, and the sensitivity and specificity were 92.3% and 81.5%, respectively. AUC of CCND2 level in the diagnosis of thyroid cancer was 0.837, and the sensitivity and specificity were 67.7% and 89.2%, respectively. The AUC of combined detection of CCND2 and miR-206 in the diagnosis of thyroid cancer was 0.959, and the sensitivity and specificity were 93.8% and 87.7%, respectively. The levels of miR-206 and CCND2 were significantly correlated with TNM staging and lymph node metastasis. CONCLUSIONS: miR-206 and CCND2 may become new biomarkers for clinical diagnosis of thyroid cancer based on the fine-needle aspiration cytology of thyroid nodules.

FBXL6 is dysregulated in keloids and promotes keloid fibroblast growth by inducing c-Myc expression.

C-MYC-mediated keloid fibroblasts proliferation and collagen deposit may contribute to the development of keloids. F-box and leucine-rich repeat protein 6 (FBXL6) is reported to be involved in tumour progression, while the role of FBXL6 in keloid fibroblasts is not deciphered. Normal control skins, hypertrophic scars and keloid tissues were collected and prepared for FBXL6 detection. FBXL6 short hairpin RNAs (shRNAs) or FBXL6 over-expression plasmids were transfected into keloid fibroblasts, and then c-MYC plasmids were further transfected. Cell viability was assayed with a Cell-Counting Kit-8 kit. The relative expression of FBXL6, Cyclin A1, Cyclin D2, Cyclin E1 and Collagen I was detected with real-time PCR and Western blot. Elevated FBXL6 expression could be observed in keloid tissues and hypertrophic scars. FBXL6 shRNAs transfection could inhibit the viability of keloid fibroblasts with diminished c-MYC expression and down-regulated Cyclin A1, Cyclin D2, Cyclin E1 and Collagen I expression. At the same time, overexpressed FBXL6 could promote the proliferation of keloid fibroblasts. Overexpression of c-MYC could promote the proliferation of keloid fibroblasts reduced by FBXL6 shRNAs with up-regulated Cyclin A1 and Collagen I expression. FBXL6 could promote the growth of keloid fibroblasts by inducing c-MYC expression, which could be targeted in keloids treatment.

[LncRNA RPL22P1-201 affects prostate cancer cell proliferation, cell cycle, and sensitivity to docetaxel by regulating miR-216b-5p expression].

OBJECTIVE: Exploring the effects and mechanisms of long non coding RNA (lncRNA) RPL22P1-201 on prostate cancer cell proliferation, cell cycle, and docetaxel sensitivity by regulating miR-216b-5p expression. METHODS: The Cancer LncRNA Census database was used to analyze the differential expression of RPL22P1-201 between prostate cancer tissue and normal tissue. Real time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression level of RPL22P1-201 in prostate cancer cell lines (DU-145, C4-2B, PC3, 22Rv1, LNCaP) and normal prostate epithelial cells (RWPE-1). PC3 cells were divided into si-RPL22P1-201 group (transfected with RPL22P1-201 interference sequence) and si-NC group (transfected with si-NC sequence). Colony formation assay was used to detect the proliferation ability of PC3 cells. Flow cytometry was used to detect the PC3 cell cycle. The CCK-8 method was used to detect the proliferation of PC3 cells in each group after treatment with docetaxel. The dual luciferase reporter gene experiment verifies the binding of RPL22P1-201 to the target gene. qRT-PCR was used to detect the expression level of miR-216b-5p. Western blot was used to detect the expression levels of TrkB, CDK4, cyclin D2, cyclin D3, and CDK6 proteins. RESULTS: The expression level of RPL22P1-201 in prostate cancer tissue was higher than that in normal tissue (P<0.01). The expression level of RPL22P1-201 in prostate cancer cell lines was higher than that in normal prostate epithelial cells (P<0.01). The number of colonies in the si-NC group and si-RPL22P1-201 group was (256.1 ± 28.79) and (78.77 ± 14.52), respectively. The difference was statistically significant (P<0.01). The G0/G1 cell rates in the si-NC group and si-RPL22P1-201 group were (43.18 ± 4.56)% and (68.85 ± 3.40)%, respectively. The S cell rates were (36.84 ± 2.28)% and (24.27 ± 2.74)%, respectively. The G2/M cell rates were (19.98 ± 2.69)% and (6.88 ± 1.57)%, respectively, and the differences were statistically significant (all P<0.05). The cell survival rate of the si-RPL22P1-201 group under the action of docetaxel was lower than that of the si-NC group (all P<0.05). RPL22P1-201 can pair and bind with miR-216b-5p (P<0.01). Compared with the si-NC group, the si-RPL22P1-201 group showed a decrease in miR-216b-5p expression in PC3 cells (P<0.01), and a decrease in TrkB, CDK4, cyclin D2, cyclin D3, and CDK6 protein expression. CONCLUSIONS: RPL22P1-201 is highly expressed in prostate cancer, and silencing RPL22P1-201 inhibits prostate cancer PC3 cell proliferation and cell cycle by increasing miR-216b-5p expression, and enhances PC3 cell sensitivity to docetaxel.