The oncomiR miR-197 is a novel prognostic indicator for non-small cell lung cancer patients.

BACKGROUND: MicroRNA expression signatures can promote personalised care for non-small cell lung cancer (NSCLC) patients. Our aim was to evaluate the previously unexplored prognostic potential of miR-197, a key oncogenic molecule for NSCLC. METHODS: Total RNA isolation (n=124 NSCLC and n=21 tumour-adjacent normal tissues), was performed using the QIAsymphony SP workstation. The quantity and quality of RNA were assessed by spectrophotometric analysis and an Agilent 2100 bioanalyzer. Polyadenylation and reverse transcription were subsequently carried out. MiR-197 expression levels were measured by qPCR, after quality control (inter-assay CV=7.8%). Internal validation procedures were followed by assigning training and test sets and robust biostatistical analyses were performed, including bootstrap resampling. RESULTS: MiR-197 is associated with larger tumours (P=0.042) and the squamous cell carcinoma histotype (P=0.032). Interestingly, after adjusting for important prognostic indicators, miR-197 expression was identified as a novel independent predictor of unfavourable prognosis for NSCLC patients (HR=1.97, 95% CI=1.10-3.38, P=0.013). We also demonstrate that miR-197 retains its prognostic performance in both early-stage I (P=0.045) and more advanced-stage individuals (P=0.036). CONCLUSIONS: The cost-effective expression analysis of miR-197 could constitute a novel molecular tool for NSCLC management.

Human kallikrein-related peptidase 12 stimulates endothelial cell migration by remodeling the fibronectin matrix.

Kallikrein-related peptidase 12 (KLK12) is a kallikrein family peptidase involved in angiogenesis - a complex biological process in which the sprouting, migration and stabilization of endothelial cells requires extracellular matrix remodeling. To characterize the molecular mechanisms associated with KLK12's proangiogenic activity, we evaluated its ability to hydrolyze various matrix proteins. Our results show that KLK12 efficiently cleaved the human extracellular matrix proteins fibronectin and tenascin, both of which are involved in the regulation of endothelial cell adhesion and migration. For fibronectin, the major proteolytic product generated by KLK12 was a 29 kDa fragment containing the amino-terminal domain and the first five type I fibronectin-domains, which are essential for regulating fibronectin assembly. We also demonstrated that KLK12-mediated fibronectin proteolysis antagonizes fibronectin polymerization and fibronectin fibril formation by endothelial cells, leading to an increase in cell migration. Furthermore, a polyclonal antibody raised against KLK12's proteolytic cleavage site on fibronectin prevented the KLK12-dependent inhibition of fibronectin polymerization and the KLK12-mediated pro-migratory effect on endothelial cells. Taken as a whole, our results indicate that KLK12's proangiogenic effect is mediated through several molecular mechanisms.

Molecular cloning and expression of an alternative hKLK3 transcript coding for a variant protein of prostate-specific antigen.

We report the molecular cloning of a full-length cDNA corresponding to a 2.1-kb hKLK3 mRNA. This mRNA results from the alternative splicing of intron 4, and its accumulation in prostatic LNCaP cells is stimulated by androgen. The cDNA encodes a prepro-prostate-specific antigen (PSA) variant containing 238 amino acids. The new protein, PSA-related protein 1 (PSA-RP1), differs from PSA at the COOH-terminal end and lacks the serine residue that is essential for catalytic activity. Prepro-PSA-RP1 was transiently expressed in COS1 cells fused to the V5 epitope of the paramyxovirus SV5. The recombinant fusion protein was detected in the spent medium by Western blot analysis using anti-V5 and anti-PSA antibodies. This indicates that PSA-RP1 is secreted and has PSA-like antigenic epitopes. A pro-PSA and a pro-PSA-RP1 having a mutated propeptide were overproduced in Escherichia coli fused to glutathione S-transferase. The recombinant PSA and PSA-RP1 were matured in vitro and identified by Western blot with molecular masses of 29 (PSA) and 27 (PSA-RP1) kDa. The data indicate that PSA-RP1, not complexed to serine protease inhibitors, could be present in biological fluids, thus contributing to the free PSA-immunoreactive fraction in serum.

Purification and characterization of active recombinant rat kallikrein rK9.

The rat tissue kallikrein rK9 is most abundant in the submandibular gland and the prostate. It has been successfully expressed in the Pichia pastoris yeast expression system. A full-length cDNA coding for the mature rK9 was fused in frame with yeast alpha-factor cDNA. The fusion protein was secreted into the medium with high yield without being processed by the yeast KEX2 signal peptidase. Mature rK9 was efficiently released from the fusion protein by trypsin and was purified to homogeneity by one-step affinity chromatography using soya bean trypsin inhibitor (SBTI) as affinity ligand. The identity of the recombinant enzyme was checked by N-terminal amino acid sequencing, Western blot analysis and kinetic studies. The dual trypsin- and chymotrypsin-like enzymatic specificity of rK9 was assessed by determining specificity constants (k(cat)/K(m)) for the hydrolysis of fluorogenic substrates, the peptide sequences of which were derived from proparathyroid hormone (pro-PTH) and from semenogelin-I. Our results confirmed the presence of an extended binding site in the rK9 active site. We also identified a far more sensitive substrate of this enzyme than those previously described, Abz-VKKRSARQ-EDDnp, which was hydrolysed with a catalytic efficiency k(cat)/K(m) of 420000 M(-1)s(-1). Finally, we showed that four of the five major proteins contained in secretions of rat seminal vesicles were rapidly degraded by recombinant rK9.

Various transcripts are generated from the VCSA1 gene by alternative splicing and poly(A) processing in the rat submandibular gland.

The members of the VCS (variable coding sequence) multigene family display extensive evolutionary divergence in the protein-coding region. The first described gene (VCSA1) was found to encode a major 0.7-kb mRNA (VCSA1*1T1) coding for a prohormone-like preproprotein, SMR1-VA1, in the submandibular gland (SMG) of Rattus norvegicus. We report here the cloning of four other VCSA1 cDNAs corresponding to mRNAs (VCSA1*1T2 to *1T5) expressed in the SMG. VCSA1*1T1 to *1T4 mRNAs share the three exons previously described and differ in their 3' untranslated regions (UTR). Their differences originate from the alternative utilization of four polyadenylation sites. Comparison of the tissue levels of VCSA1*1T1 and VCSA1*1T4 during post-natal development of the male rat SMG suggests that the poly(A) addition sites are both used at each stage. The fifth RNA transcript (VCSA1*1T5) contains only the first two exons. The nucleotide sequence of the cDNA reveals that VCSA1 has an additional exon (exon 4) which is spliced to exon 2 in VCSA1*1T5. In addition to VCSA1*1T1, at least VCSA1*1T4 and VCSA1*1T5 are actively translated in vivo, as revealed by their association to the polysomal fractions. The protein, P2-VA1, coded by VCSA1*1T5 is 68 amino acids in length and it is likely to be a glycosylated secretory protein. The putative mature P2-VA1 protein completely differs from the SMR1-VA1 pro-protein and very likely has a different function. VCSA1*1T1 is accumulated in the male rat SMG 200-1000-fold more than the other transcripts. Run-on experiments reveal that almost all transcription proceeds several hundred bp downstream from the poly(A) site corresponding to VCSA1*1T1.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of mRNAs of two androgen-dependent proteins, SMR1 and SMR2, by in situ hybridization reveals sexual differences in acinar cells of rat submandibular gland.

Androgen-dependent sexual differences in the granular convoluted tubules of mouse and rat submandibular glands (SMG) have been extensively reported. We studied two major androgen-dependent mRNAs of the rat SMG encoding proteins named SMR1 and SMR2. To determine which cell type in the SMG is responsible for synthesis of these mRNAs, we performed in situ hybridization with digoxigenin-labeled RNA probes coupled with alkaline phosphatase detection. We show that SMR1 and SMR2 mRNAs are synthesized in the acinar cells of the SMG. A clear difference in SMR1 and SMR2 mRNA levels in male and female is demonstrated. During the course of this study we also confirmed the acinar localization of mRNAs encoding the glutamine/glutamic acid-rich proteins (GRP) of rat SMG. Our data are the first clear evidence of androgen-dependent sexual differences in acinar cells of rat submandibular gland.

Molecular cloning of two cDNAs for related secretory proteins in lizard epididymis: gene expression during androgen-induced cell growth and secretion.

Lizard epididymis is an androgen-dependent tissue which produces notably ten related secretory proteins (L-proteins, Mr 19,000) during the reproductive period. These proteins were synthesized in vitro as preproteins (Mr 25,000, 24,000, 23,500). A cDNA library in the plasmid pBr322 was constructed and two cDNA clones were isolated by differential hybridization according to the differential expression of the mRNAs in stages 1 and 6 of the annual reproductive cycle. Translations of mRNAs hybrid-selected by two clones (LV123, LV132) yielded proteins which were immunoprecipitated by the L-antiserum. These preproteins were processed in vitro into six peptides; four were encoded by mRNAs selected with the LV123 clone, the others by the LV132 clone. Only three bands were detected using Northern blot analysis suggesting that the L-family could be derived from various mRNAs and from post-translational maturations. Southern analysis of genomic DNA suggests that the L-mRNAs were encoded by at least two distinct genes which could exist in numerous copies. The L-gene expression was studied under various physiological conditions and was found to be androgen-dependent. Furthermore, the results suggest the presence of a translational regulation in the newly differentiated epithelial cells.

Recent evolution of genes encoding the prohormone-like protein SMR1 in the rat submandibular gland.

The Variable Coding Sequence (VCS) multigene family of Rattus norvegicus, is composed of at least 10 members, and shows extensive evolutionary divergence in the protein-coding region. Three members of the VCSA subclass, have been characterized: one of them, the VCSA1 gene mainly expressed in the submandibular gland (SMG) encodes the prohormone-like protein, SMR1-VA1. As VCSA-related genes have not been detected in Mus musculus, the VCSA genes subclass is presumed to have recently emerged. To study the evolution of this subclass, we have looked for VCSA genes in a closely related species, Rattus rattus. By Northern analysis, we demonstrate that VCS-related mRNAs are present in the SMG, and that the level of VCSA mRNA accumulation is approximately equal in both sexes. By contrast, in R. norvegicus, males accumulate about 3,000 times more VCSA1 mRNA than females. Using total SMG mRNA, an almost full-length cDNA, homologous to the cDNA of the R. norvegicus VCSA1 gene, was cloned by reverse transcriptase polymerase chain reaction (RT-PCR). The putative corresponding SMR1-VA1 protein is 146 amino acids long and presents the features characteristic of a secreted protein, with a potential signal peptide of 22 amino acids in the amino-terminal portion. The presence of potential processing multibasic sites suggests that small peptides could be generated (particularly a hexapeptide: Arg-Gln-His-Asn-Leu-Arg), as in the case of the SMR1-VA1 protein of R. norvegicus. From Southern blot analysis there appears that species-species modifications of VCSA gene copy number have occurred; R. rattus contains a greater VCSA1 copy number than R. norvegicus (two or three and one, respectively).

Testosterone and corticosterone co-regulate messenger RNA coding for secretory proteins in the epididymis of the lizard (Lacerta vivipara).

The hormonal requirements for the regulation of Lv132 mRNA coding for two proteins secreted by the principal cells of the lizard epididymis were examined by organotypic culture experiments. Testosterone, R1881 and corticosterone induced accumulation of Lv132 mRNA in explants from lizards castrated immediately after differentiation of the principal cells. The induction by testosterone was inhibited by the addition of cyproterone acetate. Progesterone and oestradiol alone or in presence of testosterone were ineffective. Unlike the induction by testosterone, the effect of corticosterone did not require binding on the androgen receptor as shown by competition binding studies. Corticosterone failed to induce gene expression in organs containing only reserve cells in their epithelium at the onset of the culture. However, corticosterone plus testosterone had a synergistic effect. These data suggest that testosterone promotes the differentiation of principal cells from reserve cells during the culture time and that a primary action of testosterone is necessary to confer corticosterone responsiveness on this tissue. Furthermore, the primary effects of testosterone could be memorized by the tissue because the corticosterone responsiveness persists after castration.

[Simultaneous radioimmunoassay of androstenedione, testosterone and dihydrotestosterone. Application to the study of Leydig cell function]. / Dosage simultané, par radioimmunologie, de l'androstènedione, de la testostérone et de la dihydrotestostérone. Application à l'étude du fonctionnement des cellules de Leydig.

A radioimmunological method for simultaneous dosage of androstenedione, testosterone and dihyhydrotestosterone is described. This technique was applied to the study of the secretions of Leydig cells removed from hypophysectomized boar testis, in organ cultures. The releasing kinetic of the 3 steroids under hCG influence was studied; after 12 days of culture, the medium was analysed during 96 hours (8 periods of 12 hours); the steroid production started immediately and showed a progressive increase then slightly slowed down at the end of the 4 days.

[Testicular androgens and spermatogenetic cycles in the viviparous lizard]. / Androgènes testiculaires et cycles spermatogénétiques chez le lézard vivipare.

Androstenedione, testosterone and dihydrotestosterone levels were measured in the testis of 36 males of the viviparous lizard throughout a period (from end of May to end of July) characterized by the transition between two spermatogenetic cycles and by very low levels of plasma testosterone. The sudden rise of testicular testosterone and androstenedione in June is concomitant with a degeneration of the seminiferous epithelium. It coincides with a transient appearance of testicular dihydrotestosterone. During the next decline in the levels of testosterone and androstenedione, it occurs a restoration of the seminiferous tubules which resume spermatogenesis (proliferation of spermatogenia and prophase of first meiotic division). The part played by some testicular steroids in the control of spermatogenesis is discussed.

Effects of differentiation state and post-castration time lapse on the epididymal response of the lizard to testosterone in vitro: changes in specific protein and mRNA levels.

The epididymis of the viviparous lizard secretes large amounts of proteins among which L-proteins are prominent components. It undergoes great morphological and physiological modifications during its testosterone-controlled annual cycle. The effects of testosterone on L-proteins synthesis and L-mRNA concentrations were studied in cultures of organs regressed after castration. Of three tested serum supplements (2% Ultroser, 10% fetal calf serum, 10% calf serum) calf serum was shown to be essential for androgen-specific control of L-proteins synthesis. The duration of castration governed the in-vitro response to testosterone principally at the level of L-proteins synthesis. The onset of synthesis was delayed in 2-month post-castration explants, compared with 1-month post-castration explants, and was dissociated from appearance of the mRNA. This suggests that there is translational control of secretory proteins in the regressed epididymis. Conversely, the response to testosterone at the mRNA level was delayed in explants from animals castrated during a non-secretory state, compared with explants from animals castrated at the onset of secretion. These results, together with other data, suggest that expression of the L-proteins is under multifactorial control and that the influence of multiple controlling elements varies with the stage of differentiation.

Characterization and androgenic regulation of major mRNAs coding for epididymal proteins in a lizard (Lacerta vivipara).

During the reproductive period (spring) under the control of testosterone the epididymis of the viviparous lizard secretes a group of major proteins with an approximate Mr of 19,000 named L protein(s). These proteins are recognized by a specific immunoserum and bind to the heads of spermatozoa. During spring, translation in reticulocyte lysate of RNA from secreting epididymis (stage 6) produced 5 immunoprecipitable bands with Mr values from 21,500 to 25,000. Such synthesis is undetectable during sexual rest in summer (stage 1). The 5 bands disappear when translation is performed in the presence of dog pancreas microsomes although a new band of Mr 19 000 becomes prominent. This suggests that synthesis of L protein involves two steps, i.e. synthesis of precursors (L preproteins) followed by a maturation process. At least 11 translation products (including L-preproteins) are involved in annual variations that follow the differentiation of the epididymal epithelial cells and their androgen dependency was studied by castration and in-vitro stimulation by testosterone. In these conditions, testosterone is able to control accumulation of RNA corresponding to L preproteins and to a translation product of Mr 29 000.

Changes in epididymal protein synthesis during the sexual cycle of the lizard, Lacerta vivipara.

During its annual cycle, the lizard epididymis undergoes strong modifications of the secretory epithelium. These modifications previously were classified into 10 stages. The present study gives the biochemical basis of these modifications. Several parameters, such as the quantity of soluble proteins, rates of protein synthesis, and electrophoretic profiles of newly synthesized proteins and of in vitro RNA translation products were compared at 8 stages. Two-dimensional gel electrophoresis of newly synthesized tissue proteins showed that the synthesis of about 20 proteins fluctuated during the cycle. Furthermore, it revealed that the protein band L of molecular weight 19,000 identified in one-dimensional (1-D) electrophoresis was composed of at least 10 proteins. Their rate of synthesis paralleled the concentrations of their mRNA evaluated with in vitro translation. This could indicate that in this system protein synthesis is regulated by mRNA concentrations. The present analysis has confirmed that 4 different phases characterize the annual evolution of the lizard epididymis: regeneration, onset of secretory activity, hypersecretion and involution. Well-defined, newly synthesized proteins would characterize some of these phases, and could be used as markers for future detailed analysis of epididymis control.

Episodic evolution and rapid divergence of members of the rat multigene family encoding the salivary prohormone-like protein SMR1.

In rodents, the variable coding sequence (VCS) multigene family displays extensive evolutionary divergence in the protein-coding region. While certain VCS genes coding for proline-rich proteins (hPR-PB, mMSG1, rPR-VB1) are conserved in primates and rodents, others seem to be specific to certain genera. This appears to be the case for the Rattus genes forming the A-subclass. This subclass is composed of three genes in R. norvegicus and probably five genes in R. rattus. The first described VCSA gene (Rn. VCSA1) was found to encode a prohormone-like protein named SMR1 (-VA1), expressed mainly in the submandibular glands (SMG) of male rats. To further understand the evolution of this variable multigene family, we have cloned the two additional genes (Rn. VCSA2 and Rn. VCSA3) forming the R. norvegicus A-subclass and three VCSA genes (Rr. VCSA1a, b and Rr. VCSA2) of R. rattus. The putative SMR1 proteins encoded by all these genes display the same prohormone-like structure as Rn. SMR1-VA1. However, we observe a polymorphism in some internal cleavage sites which suggests that multiple processing of the SMR1 proteins could result in the liberation of peptides differing in structure and length. The phylogenetic analysis of the sequences reveals that the duplication events giving rise to the VCSA1, -A2, and -A3 progenitors were anterior to the R. norvegicus and R. rattus split, and that a VCSA1 duplication event likely occurred specifically in R. rattus. A striking observation is that the coding sequences of the VCSA genes have rapidly diverged from their ancestors. Along all branches of the phylogeny, the nonsynonymous divergence rate is identical or superior to the synonymous divergence rate. We suggest that frequent changes in functional requirements are mainly responsible for the episodic evolution and the rapid divergence of the VCSA genes.

A new proline-rich protein precursor expressed in the salivary glands of the rat is encoded by a gene homologous to the gene coding for the prohormone-like protein SMR1.

A gene encoding a prohormone-like protein (SMR1) has previously been characterized and shown to be expressed in the rat submandibular glands under androgenic control (Rosinski-Chupin, I., Tronik, D., and Rougeon, F. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8553-8557). This gene, now named VCS-alpha 1, belongs to a multigene family (Rosinski-Chupin, I., and Rougeon, F. (1990) DNA Cell Biol. 9, 553-559). We now describe the structure and the expression of a second member (VCS-beta 1) of this family. The two genes differ principally in the protein-coding region, therefore we have named these related genes VCS (variable coding sequence). Genomic clones containing the VCS-beta 1 gene were obtained by screening a lambda EMBL3 library with a probe corresponding to the VCS-alpha 1 cDNA. The nucleotide sequence of VCS-beta 1 predicted a structure containing three exons. This structure, confirmed by sequencing a VCS-beta 1 cDNA obtained by reverse polymerase chain reaction, is identical to the organization of the VCS-alpha 1 gene. Comparison of the VCS-beta 1 and VCS-alpha 1 genomic sequences indicates regions of homology which are unevenly distributed, suggesting a differential evolution of some areas (particularly the third exon) of the VCS genes. The VCS-beta 1 cDNA codes for a proline-rich protein precursor named PR-V beta 1 (148 amino acids, 39.2% proline, 10.8% glycine) and characterized by a secretory signal-peptide and three repeats of a unit rich in proline residues surrounded by two clusters of potential endoprotease cleavage sites. mrNA coding for PR-V beta 1 was detected in the submandibular-sublingual gland complex of male and female rats. PR-V beta 1 is homologous to the proline-rich peptide B isolated from human saliva (Isemura, S., Saitoh, E., and Sanada, K. (1979) J. Biochem. (Tokyo) 86, 79-86) and to the submandibular proline-rich protein precursor MSG1 of the mouse (D. Tronik-Le Roux, M. Senorale-Pose, and F. Rougeon, manuscript in preparation). Our observations provide evidence that in addition to the known proline-rich protein genes, there is, in rodent and probably human genomes, another class of genes coding for salivary proline-rich proteins. The high conservation of various sites for bacterial collagenases localized in the repeat region of PR-V beta 1, MSG1, and PRP-B suggest a protective function of these proteins in the oral cavity.

Effects of castration on the epididymis of a non-mammalian vertebrate: evolution of morphology, protein synthesis, and of specific mRNA levels.

The lizard epididymis is an androgen-dependent organ undergoing large variations in its structure and in protein synthesis ability during its annual cycle. It produces a major androgen-dependent protein, the L protein. This work reports the effects of castration performed at 3 prominent points of the sexual cycle: stage 1 (epithelium reorganization) stage 3 (onset of secretory activity) stage 6 (full secretory activity). Evolution of various parameters (organ weight, histology, amount of soluble proteins, rate of soluble protein synthesis and of specific protein synthesis: L protein, and mRNA levels) was considered over a period ranging from 7 days to 15, 30 and 60 days. Deprivation of the testis was followed by an organ involution which was more or less accentuated or more or less rapid according to the stage of the operation but some peculiarities need to be emphasized. At first, the evolution of the organ was not stopped but it proceeded: at stage 1, there was cell division and a correlated increase in total protein synthesis (without L protein synthesis), at stage 3 total protein synthesis and L protein mRNA levels increased (synthesis of L protein proceeded up to 30 to 60 days), at stage 6 the involution was accelerated. These effects concerning stage 1 and particularly stage 3 appeared as a kind of a paradoxical induction. Secondly, the epithelium underwent phases of destruction and regeneration which were obviously not controlled by the testis.

[In vitro translation of RNA from lizard epididymis and identification of poly A RNA coding for a major androgen-dependent protein]. / Traduction in vitro d'ARN d'épididyme de lézard et identification d'un ARN poly A codant pour une protéine majeure androgéno-dépendante.

During the reproductive period (spring) the lizard epididymis secretes a soluble protein of an apparent molecular weight (MW) of 19,000, the protein L. This androgen dependent protein disappears during post-nuptial atrophy of the epididymis (summer). At two time intervals (spring and summer) total RNA were extracted and poly (A) RNA were prepared. The RNA were translated in a cell-free system (rabbit reticulocyte lysate) in the presence of 35S methionine. Labelled translation products were analyzed by polyacrylamide gel electrophoresis under denaturing conditions. Electrophoresis were preceded or not by immunoprecipitation with an antiserum raised against protein L. RNA extracted during spring coded for several unique bands including five immunoprecipitated proteins with close-related MW (21,000 to 25,000). When RNA were translated in the presence of dog microsomes, the five previously detected protein bands disappear although a 19,000 MW immunoprecipitated protein was clearly demonstrated. These proteins were not detected when RNA extracted in summer were used. The protein L appears to be synthesized as preprotein(s). Its (unique or several?) messenger is of poly A type; it is present in spring and absent or undetectable in these experimental conditions in summer.

Evolution and testosterone content of the epididymis during the annual cycle of the lizard Lacerta vivipara.

The lizard epididymis provides a model for studying the control, by testosterone, of a secretory activity related to the physiology of spermatozoa. To evaluate seasonal changes and to establish chronological correlations between the structure of the epididymis and its testosterone content, lizards (Lacerta vivipara) were killed between March (emergence) and October (retreat). The epididymal tissue was examined histologically and assayed for testosterone content. Ten stages of development were defined, mainly on the basis of the epithelial structure and the morphological features of secretory activity. Degeneration of the epithelium after the breeding period and its subsequent renewal also were considered. Increased epithelial height and secretory activity coincided with a progressive rise of the testosterone level, and a severe atrophy followed a sudden reduction of blood testosterone. Reorganization of the epithelium takes place when testosterone is at its lowest level, and the hormonal dependency of this stage is questionable. This study confirms in vivo, during a sexual cycle, experimental evidence previously obtained concerning testosterone's control of the secretory activity of the lizard epididymis.