MiR-328-3p inhibits lung adenocarcinoma-genesis by downregulation PYCR1.

BACKGROUND: A vast majority of patients with NSCLC (non-small cell lung cancer) have lung adenocarcinoma (LA), and the survival rate of LA varies from 5% to 75% depending on the severity of this adenocarcinoma. PYCR1 (abnormal pyrroline-5-carboxylate reductase 1) gene and miR-328-3p have been found to be associated with cancer development. However, the underlying mechanism of interaction between miR-328-3p and PYCR1 in LA needs further investigation. METHODS: The expressions of miR-328-3p and PYCR1 in samples with LA were identified by RT-qPCR. Next, we investigated the targeting relationship between these two biological factors using luciferase assay. CCK-8, BrdU, transwell-migration, and flow cytometry assays were performed to detect cell viability, cell proliferation, cell migration and cell apoptosis in LA cells. RESULTS: We noticed that miR-328-3p expression was downregulated in LA samples. MiR-328-3p mimic restricted cell proliferation and cell migration, while it enhanced cell apoptosis. Furthermore, the overexpression of PYCR1 promoted the proliferation and migration of LA cells, but it repressed cell apoptosis. Moreover, PYCR1 directly interacted with miR-328-3p in the LA cells, and miR-328-3p restrained the expression of PYCR1, thus suppressing LA tumorigenesis. CONCLUSION: In summary, our study revealed that miR-328-3p targeting to PYCR1 suppressed the malignancy of LA cells by impeding cell proliferation and migration, while effectively promoting cell apoptosis.

PYCR1 promotes the progression of non-small-cell lung cancer under the negative regulation of miR-488.

PYCR1 is over-expressed in non-small-cell lung cancer (NSCLC) and its high expression accelerates the progression of NSCLC. However, the underlying mechanisms of PYCR1 in NSCLC progression remain poorly understood. Our study determined the mechanisms of PYCR1 in promotion of the occurrence and development of NSCLC in vitro and in vivo. Firstly, the expression patterns of PYCR1 in NSCLC tissues and cells were determined by RT-PCR, western blot and immunohistochemistry. Then, the effects of PYCR1 on cell proliferation and apoptosis were evaluated by CCK-8 and flow cytomery assays. Finally, we explored the up-regulatory microRNAs (miRs) of PYCR1 and determined if MAPK pathway involved in this process. PYCR1 expression was elevated in NSCLC tissue samples and cells, and the high expression of PYCR1 closely associated with patients' advanced clinical process and poor outcome. Up-regulation of PYCR1 significantly increased the expression of p38 and promoted its nuclear accumulation. Besides, PYCR1 expression was negatively regulated by miR-488, and up-regulation of miR-488 significantly inhibited cell proliferation and tumorigenesis and increased cell apoptosis, and decreased p38 expression and its nuclear accumulation, whereas up-regulation of PYCR1 rescued these results induced by miR-488 over-expression. Collectively, these data suggest the mechanism of PYCR1 in promotion of NSCLC progression. PYCR1 is negatively regulated by miR-488 and then promotes the occurrence and development of NSCLC and activates p38 MAPK pathway.

Knockdown of PYCR1 inhibits cell proliferation and colony formation via cell cycle arrest and apoptosis in prostate cancer.

Pyrroline-5-carboxylate reductase 1 (PYCR1) is an enzyme involved in cell metabolism, which has been shown to be up-regulated in cancers. However, the functions of PYCR1 in prostate cancers (PCa) are still largely unknown. In the present study, we found that PYCR1 was highly expressed in prostate cancer tissues and then knocked down PYCR1 in PCa cell lines (DU145, PC-3 and LNCap) via lentivirus-mediated gene delivery and analyzed its biological function. Both qRT-PCR and western blotting indicated that PYCR1 was suppressed efficiently after sh-PYCR1 infection. Further analysis indicated knockdown of PYCR1 significantly inhibited PCa cell growth and colony formation ability. The inhibition effects on growth were likely due to G2/M-phase arrest and enhanced cell apoptosis, as determined by flow cytometer analysis. At last, we verified the expression levels of cell cycle regulatory proteins, including CDK1, CDK2, CDK4 and Cyclin B1 were all downregulated and cell apoptotic-related proteins, including cleaved caspase 3 and cleaved PARP were increased in PCa cells after PYCR1 knockdown. Furthermore, PYCR1 has been shown not to be directly regulated by androgen receptor (AR) levels. These results show the functions of PYCR1 in PCa tumorigenesis for the first time and suggest that PYCR1 might be a good potential therapy approach for treating PCa.

PYCR2 Mutations cause a lethal syndrome of microcephaly and failure to thrive.

OBJECTIVE: A study was undertaken to characterize the clinical features of the newly described hypomyelinating leukodystrophy type 10 with microcephaly. This is an autosomal recessive disorder mapped to chromosome 1q42.12 due to mutations in the PYCR2 gene, encoding an enzyme involved in proline synthesis in mitochondria. METHODS: From several international clinics, 11 consanguineous families were identified with PYCR2 mutations by whole exome or targeted sequencing, with detailed clinical and radiological phenotyping. Selective mutations from patients were tested for effect on protein function. RESULTS: The characteristic clinical presentation of patients with PYCR2 mutations included failure to thrive, microcephaly, craniofacial dysmorphism, progressive psychomotor disability, hyperkinetic movements, and axial hypotonia with variable appendicular spasticity. Patients did not survive beyond the first decade of life. Brain magnetic resonance imaging showed global brain atrophy and white matter T2 hyperintensities. Routine serum metabolic profiles were unremarkable. Both nonsense and missense mutations were identified, which impaired protein multimerization. INTERPRETATION: PYCR2-related syndrome represents a clinically recognizable condition in which PYCR2 mutations lead to protein dysfunction, not detectable on routine biochemical assessments. Mutations predict a poor outcome, probably as a result of impaired mitochondrial function. Ann Neurol 2016;80:59-70.

Identification of novel androgen receptor target genes in prostate cancer.

BACKGROUND: The androgen receptor (AR) plays critical roles in both androgen-dependent and castrate-resistant prostate cancer (PCa). However, little is known about AR target genes that mediate the receptor's roles in disease progression. RESULTS: Using Chromatin Immunoprecipitation (ChIP) Display, we discovered 19 novel loci occupied by the AR in castrate resistant C4-2B PCa cells. Only four of the 19 AR-occupied regions were within 10-kb 5'-flanking regulatory sequences. Three were located up to 4-kb 3' of the nearest gene, eight were intragenic and four were in gene deserts. Whereas the AR occupied the same loci in C4-2B (castrate resistant) and LNCaP (androgen-dependent) PCa cells, differences between the two cell lines were observed in the response of nearby genes to androgens. Among the genes strongly stimulated by DHT in C4-2B cells--D-dopachrome tautomerase (DDT), Protein kinase C delta (PRKCD), Glutathione S- transferase theta 2 (GSTT2), Transient receptor potential cation channel subfamily V member 3 (TRPV3), and Pyrroline-5-carboxylate reductase 1 (PYCR1)--most were less strongly or hardly stimulated in LNCaP cells. Another AR target gene, ornithine aminotransferase (OAT), was AR-stimulated in a ligand-independent manner, since it was repressed by AR siRNA knockdown, but not stimulated by DHT. We also present evidence for in vivo AR-mediated regulation of several genes identified by ChIP Display. For example, PRKCD and PYCR1, which may contribute to PCa cell growth and survival, are expressed in PCa biopsies from primary tumors before and after ablation and in metastatic lesions in a manner consistent with AR-mediated stimulation. CONCLUSION: AR genomic occupancy is similar between LNCaP and C4-2B cells and is not biased towards 5' gene flanking sequences. The AR transcriptionally regulates less than half the genes nearby AR-occupied regions, usually but not always, in a ligand-dependent manner. Most are stimulated and a few are repressed. In general, response is stronger in C4-2B compared to LNCaP cells. Some of the genes near AR-occupied regions appear to be regulated by the AR in vivo as evidenced by their expression levels in prostate cancer tumors of various stages. Several AR target genes discovered in the present study, for example PRKCD and PYCR1, may open avenues in PCa research and aid the development of new approaches for disease management.

Cloning, characterization, and expression of cDNAs encoding human delta 1-pyrroline-5-carboxylate dehydrogenase.

Delta 1-pyrroline-5-carboxylate dehydrogenase (P5CDh; EC 1.5.1.12), a mitochondrial matrix NAD(+)-dependent dehydrogenase, catalyzes the second step of the proline degradation pathway. Deficiency of this enzyme is associated with type II hyperprolinemia (HPII), an autosomal recessive disorder characterized by accumulation of delta 1-pyrroline-5-carboxylate (P5C) and proline. As an initial step in understanding the biochemistry of human P5CDh and molecular basis of HPII, we utilized published peptide sequence data and degenerate primer polymerase chain reaction to clone two full-length human P5CDh cDNAs, differing in length by 1 kilobase pair (kb). Both cDNAs have the identical 1689-base pair open reading frame encoding a protein of 563 residues with a predicted molecular mass of 62 kDa. The long cDNA contains an additional 1-kb insert in the 3'-untranslated region that appears to be an alternatively spliced intron. The conceptual translation of human P5CDh has 89% sequence identity with the published human P5CDh peptide sequences and 42 and 26% identity with Saccharomyces cerevisiae and Escherichia coli P5CDhs, respectively, as well as homology to several other aldehyde dehydrogenases. Both P5CDh cDNA clones detect a single 3.2-kb transcript on Northern blots of multiple human tissues, indicating the long cDNA containing the 3'-untranslated intron represents the predominant transcript. The P5CDh structural gene appears to be single copy with a size of about 20 kb localized to chromosome 1. To confirm the identity of the putative P5CDh cDNAs, we expressed them in a P5CDh-deficient strain of S. cerevisiae. Both conferred measurable P5CDh activity and the ability to grow on proline as a sole nitrogen source.

Osmoregulation of a pyrroline-5-carboxylate reductase gene in Arabidopsis thaliana.

In Arabidopsis thaliana (L.) Heynh. proline can account for up to 20% of the free amino acid pool after salt stress. Proline accumulation occurs in plants mainly by de novo synthesis from glutamate. The last step of the proline biosynthetic pathway is catalyzed by pyrroline-5-carboxylate (P5C) reductase. A gene (AT-P5C1) encoding this enzyme in A. thaliana has been cloned and sequenced. Expression of AT-P5C1 in Escherichia coli resulted in the complementation of a proC mutant to prototrophy. A comparison of the AT-P5C1 primary and secondary structures with those of six P5C reductase of other organisms is presented. With the exception of several functionally important amino acid residues, little conservation in the primary structure is seen; much greater similarity exists in the putative secondary structure. The AT-P5C1 protein is probably cytosolic. Under normal growth conditions, the P5C reductase mRNA level was significantly higher in roots and ripening seeds than in green tissue. A salt treatment of A. thaliana plants resulted in a 5-fold induction of the AT-P5C1 transcript, suggesting osmoregulation of the AT-P5C1 promoter region. Moreover, a time-course experiment indicated that this induction precedes proline accumulation.

Proline biosynthesis in Saccharomyces cerevisiae: analysis of the PRO3 gene, which encodes delta 1-pyrroline-5-carboxylate reductase.

The PRO3 gene of Saccharomyces cerevisiae encodes the 286-amino-acid protein delta 1-pyrroline-5-carboxylate reductase [L-proline:NAD(P+) 5-oxidoreductase; EC 1.5.1.2], which catalyzes the final step in proline biosynthesis. The protein has substantial similarity to the pyrroline carboxylate reductases of diverse bacterial species, soybean, and humans. Using RNA hybridization and measurements of enzyme activity, we have determined that the expression of the PRO3 gene appears to be constitutive. It is not repressed by the pathway end product (proline), induced by the initial substrate (glutamate), or regulated by the general control system. Its expression is not detectably altered when cells are grown in a wide range of nitrogen sources or when glycerol and ethanol replace glucose as the carbon source. The possibility that this enzyme has other functions in addition to proline biosynthesis is discussed.

A soybean gene encoding delta 1-pyrroline-5-carboxylate reductase was isolated by functional complementation in Escherichia coli and is found to be osmoregulated.

We have isolated several cDNA clones encoding delta 1-pyrroline-5-carboxylate reductase (P5CR, L-proline: NAD(P)+ 5-oxidoreductase, EC 1.5.1.2) which catalyzes the terminal step in proline biosynthesis, by direct complementation of a proC mutation in Escherichia coli with an expression library of soybean root nodule cDNA. The library was constructed in the lambda ZapII vector, converted to a plasmid library by in vivo excision of recombinant pBluescript phagemids, and used for transformation of the E. coli mutant. Complementing plasmids contained inserts of about 1.2 kb which hybridized to a 1.3 kb RNA transcript in nodules, uninfected roots and leaves. DNA sequence analysis of one full length cDNA clone showed that it encodes a 28 586 Mr polypeptide with 39% amino acid identity to the E. coli P5CR sequence. Genomic analysis showed that there are two to three copies of the P5CR gene in the soybean genome. The steady-state level of P5CR mRNA in root nodules was twice as high as in uninfected roots and about five times higher than in leaves. Subjecting young seedlings to osmotic stress by watering with 400 mM NaCl resulted in an almost six-fold increase in the level of root P5CR mRNA, suggesting that this gene may be osmoregulated.

Genetics and physiology of proline utilization in Saccharomyces cerevisiae: mutation causing constitutive enzyme expression.

A mutation resulting in inducer-independent expression of the proline-degradative enzymes was isolated in the yeast Saccharomyces cerevisiae. Strains carrying the mutation, put3, are partially constitutive for proline oxidase and delta 1-pyrroline-5-carboxylate dehydrogenase when grown on a medium lacking proline and are hyperinducible for both enzyme activities when grown on a proline-containing medium. put3 segregates as a single nuclear gene, is not linked to either of the presumed structural genes for proline oxidase and delta 1-pyrroline-5-carboxylate dehydrogenase, and does not affect proline transport. When heterozygous in diploid strains, put3 behaves neither fully dominant nor fully recessive. Endogenous induction by proline has been eliminated as a cause of the inducer-independent enzyme expression in the put3 mutant and the mutation is believed to be in a regulatory component of the proline-degradative pathway.

Genetics and physiology of proline utilization in Saccharomyces cerevisiae: enzyme induction by proline.

Proline is converted to glutamate in the yeast Saccharomyces cerevisiae by the sequential action of two enzymes, proline oxidase and delta 1-pyrroline-5-carboxylate (P5C) dehydrogenase. The levels of these enzymes appear to be controlled by the amount of proline in the cell. The capacity to transport proline is greatest when the cell is grown on poor nitrogen sources, such as proline or urea. Mutants have been isolated which can no longer utilize proline as the sole source of nitrogen. Mutants in put1 are deficient in proline oxidase, and those in put2 lack P5C dehydrogenase. The put1 and put2 mutations are recessive, segregate 2:2 in tetrads, and appear to be unlinked to one another. Proline induces both proline oxidase and P5C dehydrogenase. The arginine-degradative pathway intersects the proline-degradative pathway at P5C. The P5C formed from the breakdown of arginine or ornithine can induce both proline-degradative enzymes by virtue of its conversion to proline.

Effects of cortisol or starvation on the activities of four enzymes in small intestine and liver of the rat during development.

The small intestine of the rat, like the liver, is a tissue with high activities of arginase, ornithine aminotransferase, and pyrroline-5-carbozylate reductase. These enzymes are thought to catalyse sequential steps in the synthesis of proline. We have compared the effect of cortisol or brief starvation on the activities of these enzymes and of soluble alanine aminotransrerase in the small intestine and liver during development. In the intestine, cortisol accelerated the increase in arginase activity, reversed the normal 2-week-long post-natal decline in that of pyrroline-5-carboxylate reductase, and delayed the normal decrease, in the third week, of ornithine aminotransferase activity. Starvation of neonates for 18 h raised the activity of arginase slightly, that of pyrroline-5-carboxylate reductase significantly, and had no effect on ornithine aminotransferase activity. Cortisol did not alter the hepatic activities of pyrroline-5-carboxylate reductase in neonates but induced premature rises in the activities of arginase and ornithine aminotransferase. Short starvation did not affect the hepatic activities of any of these enzymes. Alanine aminotransferase activity in both tissues was enhanced by cortisol but not by starvation. Thus in intestine, cortisol elicited some changes in the activity of three functionally related and one unrelated enzyme while starvation evoked changes only in pyrroline-5-carboxylate reductase. Neither stimulus appears to be specific for a metabolic pathway or to trigger a coordinated onset of proline synthesis from arginine.