Mechanism of exogenous nucleic acids and their precursors improving the repair of intestinal epithelium after γ-irradiation in mice

Abstract

AIM: To clone expressed genes associated with repair of irradiation-damaged mice intestinal gland cells treated by small intestinal RNA, and to explore the molecular mechanism of exogenous nucleic acids improving repair of intestinal crypt.

METHODS: The animal mode of test group and control group was established, forty-five mice being irradiated by γ ray were treated with small intestinal RNA as test group, forty mice being irradiated by γ ray were treated with physiological saline as control group, five mice without irradiation were used as normal control, their jejunal specimens were collected respectively at 6 h, 12 h, 24 h, 4 d and 8 d after irradiation. Then by using LD-PCR based on subtractive hybridization, these gene fragments differentially expressed between test group and control group were obtained, and then were cloned into T vectors as well as being sequenced. Obtained sequences were screened against. GeneBank, if being new sequences, they were submitted to GeneBank.

RESULTS: Ninety clones were associated with repair of irradiation-damaged intestinal gland cells treated by intestinal RNA. These clones from test group of 6 h, 12 h, 24 h, 4 d and 8 d were respectively 18, 22, 25, 13, 12. By screening against GeneBank, 18 of which were new sequences, the others were dramatically similar to the known sequences, mainly similar to hsp, Nmi, Dutt1, alkaline phosphatase, homeobox, anti-CEA ScFv antibody, arginine/serine kinase and BMP-4, repA. Eighteen gene fragments were new sequences, their accept numbers in GeneBank were respectively AF240164-AF240181.

CONCLUSION: Ninety clones were obtained to be associated with repair of irradiation-damaged mice intestinal gland cells treated by small intestinal RNA, which may be related to abnormal expression of genes and matched proteins of hsp, Nmi, Dutt1, Na, K-ATPase, alkalineph-osphatase, glkA, single stranded replicative centromeric gene as well as 18 new sequences.

Key Words: radiation, ionizing; intestine, small/injuries; RNA; gene expression; nucleic acids/therapeutic use; polymerase chain reaction; repair; intestinal epithelium; mice

INTRODUCTION

After exposure to large dose ionizing radiations, the larger intestinal gland cell lesion is the main cause of death in humans and animals. How to enhance intestinal gland cell survival rates has become a problem to be solved. In order to increase the mouse crypt survival after irradiation, we have done a series of experiments, and finally confirmed that when a portion of the crypts was devastated by irradiation, a compensatory recovery of the intestinal epithelium by remaining crypts occurred involving three consecutive periods such as the rapid cell proliferation of the viable crypts, the fission of the proliferative crypts and the increase of crypt numbers[1]. The nucleic acid fragment containing several hundred base pairs (bp), or even any one of the nucleic acid precursors (mononucleotides, nucleosides, and bases) can enhance the crypt survival rate by 25% or so, further confirming that the effectiveness of exogenous nucleic acids depends not upon the action exerted by their highly polymerized state, but upon their various enzymatic degradation products[2]. Our experimental results suggest that the nucleic acids (DNA, RNA) and their precursors may be used as one of the effective measures for the treatment of intestinal radiation syndrome that may occur in the war time as well as in the peaceful use of atomic energy[3-5]. However, the molecular mechanism how nuclear acids and precursors improve the repairing of irradiation-damaged intestinal gland cells is unclear. In order to clarify this molecular mechanism, we used long distance-PCR based on subtractive hybridization, isolated and cloned these genes associated with repairs of irradiation-damaged mice intestinal gland cells treated by intestinal RNA. Our studies lay foundation for further clarifying molecular mechanism of repair of radiation-damaged crypt.

MATERIALS AND METHODS

Reagents

PolyATtract^® system 1000 kit from Promega was used for extraction of mRNA, SMART PCR cDNA synthesis kit (Clontech) for transcription of mRNA, Wizard^® plus Minipreps DNA purification for purification of PCR production, Advantage2PCR kit (Clontech) for LD-PCR, and PE-5700 quantiative PCR cycler used for thermal cycle. PGEM-T easy vector system was purchased from Promega, γ-³²-pdATP from Beijing Fu Rei Compancy, and the other reagents were from Beijing Yuan Ping compancy.

Establishment of the model and sample collection

Ninety BALB/c male mice with body weight of 18 g-22 g and 10-12 weeks old were randomly assigned into two groups, i.e, test group and control group. They were injected with 5 g·L^-1 barbiturate sodium 40 mg·kg^-1, put in organism radiation box, and then were irradiated on mice abdominal region by using ⁶⁰Co γ ray at the reagent rate of 149.47-151.13 cGy·min^-1, and finally reached total reagent of 1150 cGy. Small intestinal RNA was diluted into 100 mg·mL^-1. Two hours after mice being irradiated, each mouse in test group was injected 0.4 mL RNA liquid, and each mouse in control group was injected 0.4 mL physiological saline by using local intestinal cavity expanding injection method[6]. After that, the mice were raised according to conventional method. These mice were killed respectively at 6 h, 12 h, 24 h, 4 d and 8 d after irradiation, and jejunal tissues were quickly taken out, washed by physiological saline, and then were kept in liquid nitrogen. In order to avoid single difference, these samples were mixed together under identical condition.

Sample processing and LD-PCR based on subtractive hybridization

Extraction of mRNA was done according to manual of PolyATtract system 1000, and quantificated. mRNA transcription was done according to manual from SMART PCR cDNA synthesis kit. Subtractive hybridization between test group and control group was done as follows: take out 0.1 µg mRNA from the control group, add Biotinylated oligo (dT) probe (50 µmol·L^-1), 70 °C 5 min, then add into the first strand cDNA, and hybridize for 24 h at 42 °C, finally add Streptavidin magnesphere particles and mixed, magnetic steel was used to attract production of two stranded hybrids and get rid of them, and then repeat the processing twice. Take out the upper liquid, and add double volume absolute alcohol to the upper liquid to precipitate cDNAs, finally dissolve them in 10 µL Nuclease-free water. Take 10 µL of the first strand cDNA as template, sequentially add PCR-grade water 74 µL, 10 × advantage 2 PCR buffer 10 µL, 50 × dNTP mix-10 µL, 50 × advantage 2 polymerase mix 1 µL, PCR primer Mix 2 µL, 50 mmol·L^-1 MgCl₂ 3 µL, mixed well, centrifuged for several seconds, using two steps such as 95 °C,1 min, 95 °C,15 s, 68 °C, 6 min, 30 cycles, in the end elongated at 68 °C for 6 min, take out 10 µL production to run 12 g·L^-1 agarose gel electrophoresis.

Cloning and identification of PCR products

PCR products were purified using Wizard plus Minipreps DNA purification system, and dissolved in 10 µL water. According to the manual, PCR products were cloned into PGEM-T easy vector, transferred into JM109, positive clone picked up, cultured overnight, plasmid extracted out, and identified by cutting with BstZ1. Products cut by BstZ1 were labelled γ-³²pdATP at terminal, and used as probe, and hybridized with mRNA of test group and control group[7].

Sequencing and screening against GeneBank

These obtained clones were positively sequenced by using Model 377 sequencing instrument. Obtained sequences were screened against GeneBank, if being new sequences, these sequences were submitted to GeneBank[8].

RESULTS

LD-PCR based on subtractive hybridization

In test group of 6 h, 12 h, 24 h, 4 d and 8 d, products after subtractive hybridization were successfully amplified by LD-PCR, obtained bands centered on 1-1.5 kb or so (Figure 1).

Figure 1 Electrophoresis result of production of LD-PCR. 1: Marker; 2, 6: Positive control; 3, 4, 5, 7, 8: Results of 6 h, 12 h, 24 h, 4 d and 8 d; 9: Negative control

Cloning and identification of PCR products (Figure 2)

Figure 2 Results of cloning and identification of production of PCR. 1-4, 6-9: Results of part products cut by Bstz1; 5: Marker Ninety of positive clones were obtained from test group, positive clones of 6 h, 12 h, 24 h, 4 d and 8 d in test group were respectively eighteen, twenty-two, twenty-five, thirteen, twelve.

Identification of hybridization (Figure 3)

Figure 3 Part results of hybridization with RNA. A: Test group; B: Control group. Dot blots confirmed that these genes were overexpressed in test group, and lower expressed in control group, that is, these genes were associated with repair of intestinal gland cells treated by RNA.

Sequencing and searching for GeneBank

These clones were sequenced according to the results of sequencing and searching against GeneBank, eighteen were new sequences, eighty-two were dramatically similar to the known sequences.

In test group of 6 h, similar sequences mainly were as follows: mRNA for heat shock protein, Nmi mRNA, Dutt1 protein, mRNA for Na, K-ATPase gamma subunit, mRNA for surface glycoprotein, Zinc finger type transcript factor, porcine growth hormone-releasing hormone gene, monocyte/macrophage Igrelated gene, telomerase-associated protein, HOX1b protein, arginine/ serine kinase.

In test group of 12 h, similar sequences were: Alkaline phosphatase mRNA, alkaline phosphatase 2, glkA gene, single stranded replicative centromeric gene, DMBT1, tRNA-Met gene, homeobox protein, thyroxine-binding globulin gene, alpha-a-plasmin inhibitor gene.

In test group of 24 h, similar sequences were: anti-CEA ScFv antibody, anti-DNA heavy chain, mRNA for Ig kappa chain, anti BONT/A Hc ScFv antibody, mRNA for collagenase, AE0199 immunoglobulin heavy chain, Mouse Ig gamma-chain, Ig rearranged gamma-chain mRNA, anti-c-myc antibody, anti-CD30 moab ki₄ ScFv, anti-BSA antibody, D1 heavy chain, epidermal growth factor, anti-NP antibody IgH, mRNA for arginine/serine kinase.

In test group of 4 d, simliar sequences were: Dual specificity phosphatase, family mRNA telomerase-associated protein, anti-human erbB-2, tazarotene-induced gene2, betaine-GABA transporter gene, copy complex subunit 7a, mRNA for stress-activated protein, FK506 binding protein, calium/calmodulin dependent gene, PEST phosphatase interactin gene, haptoglobin mRNA, acyl-ACP desaturase, mRNA for sodium channel, peroxidase, BMP-4 gene, bone morphogenetic protein.

In test group of 8 d, similar sequences were: Ig variable region, DNA for mouse Ig, DNA for flexible peptide, tsr glkA, proteinase-3 neutroactin, EWS gene, repA protein.

Eighteen new sequences Figure 4

Figure 4 Not available

DISCUSSION

After exposing to large dose ionizing radiation, intestinal crypt radiation death occurs, and no effective therapeutic measures are available to combat it. Data showed that the devastation or death of the crypt after irradiation is the crucial factor responsible for the pathogenesis. We have performed a series of experiments intending to increase the crypt survival after-irradiation in mice and confirmed that the nucleic acids (DNA, RNA) and their precursors may be used as one of the measures for the treatment of intestinal radiation syndrome that may occur in the war as well as in the peaceful uses of atomic energy[1-5]. However, the concrete molecular and cellular mechanisms are unknown.

Human genome group comprised 100 thousand of genes, which are selectively expressed, and determined the whole life course of organism, alteration of gene expressed levels is positioned at the centrel of controlling biological adjust mechanism[8]. Therefore, we think, after irradiation, between test group treated by RNA and the control group treated by physiological saline must exist differently expressed genes, which indicate that those genes were closely associated with intestinal crypt damage and repair. To isolate and clone these genes may not only be helpful to clarify the molecular mechanism of nuclear acids treatment, but also provide important basic theory for gene therapy of irradiation damage.

In the study, using BALB/c mice as studying target, we obtained 90 of genes a ssociated with repair of irradiation damaged intestinal gland cells. Data confirmed that hsp was increased at mRNA level after chronic radiation, PARP, serine protease-like gene, p53, bcl-2, bax, argainase I, ihsr PB7, Cdx1, NPT, PCNA, D1b-1, c-Ha-ras, c-myc, c-fos and so on were also increased at mRNA levels, which were correlated closely with drug treatment of irradiation damaged intestinal cells[9-25]. In our experiment, such as Nmi mRNA, Dutt1 protein, mRNA for Na, K-ATPase gamma subunit, mRNA for surface glycoprotein, Zinc finger type transcript factor, porcine growth hormone-releasing hormone gene, monocyte/macrophage Ig-related gene, telomerase-associated protein, HOX1b protein, arginine/serine kinase, alkaline phosphatase mRNA, alkaline phosphatase 2, glkA gene et al were also closely correlated with repair of irradiation damaged intestinal crypt, what especially interesting was that RSG5 and ODC were identical to obtained sequences, data showed that RSG5, and ODC were overexpressed in irradiation-damaged intestinal crypt, and played an essential and positive role during DNA damage recovery and survival[26,27], our results also fully supported the conclusion. Although their concrete mechanism is not clarified, they may increase protein products by means of increased transcript levels to improve repair of irradiation-damaged intestinal crypt, and to suppress apoptosis of crypt cells[32].

Langberg et al[28] confirmed that immunolo-gical factors participated in the course of repair of irradiation damaged intestinal crypt such as IL-1, TGF-beta1, PDGF-AA, c-EGFR, EGF, TGF-beta-3. In our experiment, anti-CEA ScFv antibody gene, anti-DNA heavy chain, mRNA for Ig kappa chain, anti-BONT/A Hc ScFv antibody gene, mRNA for ScFv collagenase, AE0199 immunoglobulin heavy chain, mouse Ig gamma-chain, Ig rearranged gamma-chain mRNA, anti-c-myc antibody gene, anti-CD30 mAb ki-4 ScFv, anti-BSA antibody gene, D1 heavy chain, epidermal growth factor, anti-NP antibody IgH, mouse Ig gammachain and haptoglobin were likely to be correlated closely with repair of irradiation damaged intestinal crypt. What is especially interesting is several gene fragments were partly identical to sequences of ScFv genes, this point was not able to be expressed clearly. Our results support that immunological factors exert effect on the course of repair of irradiation damaged intestinal crypt[29-45].

In our experiment, eighteen novel sequences were obtained, their concrete functions are still unclear. But we believe that these genes are closely associated with irradiation treatment, only if we clarify the function of these genes, and according to the changes of these genes, to design a controlling measure, we are likely to decrease irradiation damage, and also provide new thoughts for tumor radiation treatment[46-64].

In summary, our results primarily demonstrate that nuclear acids are capable of improving repair of irradiation damaged intestinal crypt, its action may be closely correlated with increased mRNA levels of some genes, also with immunological factors, but the concrete molecular mechanism such as signal transduction and suppression of apoptosis still needs further studies[65-89].