MicroRNA-92 promotes gastric cancer cell proliferation and invasion through targeting FXR.

MicroRNAs (miRNAs), a class of small noncoding RNAs, play critical roles in human carcinogenesis through downregulation of various target genes. In the present study, we found that miR-92 is upregulated in gastric cancer tissues compared with adjacent normal tissues. Interestingly, miR-92 expression is significantly associated with clinical characteristics of patients. Gain or loss-of-function in vitro experiments further show that miR-92 mimics significantly promoted, while its antisense oligos inhibited gastric cancer cell proliferation and invasion. Moreover, luciferase reporter assays and western blot indicated that farnesoid X receptor (FXR), is a direct target of miR-92. Therefore, our data suggest that upregulation of miR-92 may represent an important mechanism for the development of gastric cancer.

[Clinical report on 68 cases of refractory mycoplasma pneumoniae pneumonia (RMPP)].

OBJECTIVE: To summarize the early diagnose and treatment approaches of Refractory Mycoplasma Pneumoniae Pneumonia (RMPP). METHODS: Medical documents of 68 cases of RMPP were reviewed. Lab and radiology evident such as CBC, CRP, MP-IgM, X-ray, etc. were collected. RESULTS: 100% RMPP patients suffered from high fever. Positive sign of lung became clear with the development of the disease. Complications as impairment of liver function, cardiac function and rush developed in few patients. 2-4 rounds treatment of macrolides and Methyllprednisolone were necessary for RMPP while antibiotic may be considered when there were evidence of bacteria infection. Immunoglobulin was recommended to the patients when macrolides and steroid seemed ineffective. Bronchofibroscope played an active role regarding the diagnosis and treatment of RMPP. CONCLUSION: Early diagnosis is crucial in RMPP. Combination of multitreatment approaches is the key to cure RMPP.

[Function of TEP1 gene during Plasmodium yoelii infection in Anopheles dirus].

OBJECTIVE: To study the role of TEP1 gene from Anopheles dirns during Plasmodium yoelii infection by RNA interference. METHODS: TEP1 primers with T7 promoter were designed based on the sequence of An. dirus TEP1 gene from GenBank database. PCR amplification of TEP1 gene was completed with An. dirus cDNA as template. The AdTEP1 double-stranded RNA was synthesized by using in vitro transcription kit with purified PCR products. Female An. dirus emerged for 1-2 days were divided into three groups each with 200 mosquitoes: TEP1 interference group, EGFP interference group and control. Mosquitoes in TEP1 and EGFP interference groups were microinjected in chest with 147 ng of AdTEP1 and EGFP double-stranded RNA, respectively, while those of control group were untreated. Effect of TEP1 interference on P. yoelii in An. dirus was estimated through semi-quantitative PCR with internal reference AdS7 at 3 d after injection. On 4 d after injection, mosquitoes were infected by EGFP-expressing P. yoelii BY265. The infection rate and infectivity of mosquitoes were observed through anastomosing 25 midguts from each group at 9 d post-infection. RESULTS: The AdTEP1 double-stranded RNA did well in the interference of TEP1 expression in An. dirus. The infection rate in the groups of control, EGFP and TEP1 interference was (24 +/- 2.83)%, (24 +/- 0.71)%, and (80 +/- 3.54)%, respectively; and the infectivity of the three groups was 0.32 +/- 0.7, 0.44 +/- 0.85, and 5.52 +/- 4.84, respectively. CONCLUSION: AdTEP1 interference increases the infection rate and infectivity of An. dirus by P. yoelii, and raises the susceptibility of An. dirus to P. yoelii significantly. TEP1 plays a critical role in the process of P. yoelii infection.

Plasmodium yoelii: correlation of TEP1 with mosquito melanization induced by nitroquine.

The antimalarial drug nitroquine is not only an effective antimalarial drug, it is also able to induce the melanization of Plasmodium species. However, the molecular mechanisms of the recognition reaction induced by this drug remain unclear. Silencing of thioester-containing protein-1 (TEP1) significantly compromised the ability of Anopheles gambiae to melanize the Plasmodium, leading to investigation of the involvement of A. stephensi TEP1 in melanization induced by nitroquine. This study shows that (1) binding of AsTEP1 to oocysts, especially melanized oocysts, (2) after ingestion of anti-AsTEP1 antibody, the melanization rate in antibody-treated mosquitoes are significantly lower than in control mosquito (p<0.05). The results suggest that nitroquine is able to induce Plasmodium recognition by TEP1, possibly triggering the resulting melanotic encapsulation. Further elucidation of the molecular mechanisms of mosquito immunity induced by antimalarial drugs will provide theoretical evidence for the use of antimalarial drugs, and a meaningful pathway for the design of novel antimalarial drugs.

[Correlation of Anopheles TEP1 gene with melanization induced by nitroquine].

OBJECTIVE: To analyze the relationship between the TEP1 gene of Anopheles stephensi and melanotic encapsulation of Plasmodium yoelii induced by anti-malaria drug nitroquine. METHODS: Haemolymph samples from three groups of An. stephensi fed with sucrose solution, Plasmodium-infected blood and nitroquine, respectively, were collected at the 1st, 2nd, 3rd and 4th day after drug administration. Degenerate primers were designed according to the conserved amino acid sequence within TEPs of the mosquitoes. Fluorescent quantitation PCR was used to detect the variation of TEP1 gene transcript induced by nitroquine. The melanization of oocysts was observed by light microscopy. RESULTS: TEP1 gene was cloned, the predicted amino acid residues harbored a highly conserved canonical thioester motif GCGEQ. The fluorescent quantitation PCR revealed that nitroquine induced an up-regulation of TEP1 activity. The transcription of TEP1 gene in nitroquine treated group (2.423) was significantly higher than that of the infected blood-fed group(1.036) at the 3rd day after nitroquine treatment (P<0.05). At the same time, most oocysts were found to be encapsulated in nitroquine treated group, while no melanized parasites were observed in the infected blood-fed group. CONCLUSION: Transcriptional variation of TEP1 gene may be related to the melanization induced by nitroquine.

Plasmodium yoelii: correlation of up-regulated prophenoloxidase and phenoloxidases with melanization induced by the antimalarial, nitroquine.

Although knowledge of the mosquito immune response has recently improved, less is known about the impact of antimalarial drugs on mosquito immunity. In the present study, we found that nitroquine, an effective antimalaria drug, could also induce melanotic encapsulation of Plasmodium by Anopheles stephensi. The melanization rate of the nitroquine treated group was 60.8%. To explore the effect of nitroquine on mosquito immunity, we determined the increase in activity of phenoloxidases (PO) enzyme, the main component of melanotic encapsulation, with nitroquine treatment. Moreover, we cloned prophenoloxidase (PPO) gene, which is accepted as the inactive phenoloxidase form and observed inducible expression of this gene with nitroquine treatment by real-time PCR. Our data implied that up-regulation of PPO gene and PO activity might be correlated with nitroquine. Nevertheless, nitroquine had no effect on the transcription of PPO gene or the activity of PO enzyme in the mosquito fed on a normal blood meal. In our study, we also observed the degenerative effect of 0.1% nitroquine on Plasmodium in the mosquito. This suggests that the degeneration of Plasmodium induced by nitroquine might result in the exposure of pattern-recognition ligands which can active the immune reaction, up-regulate PPO gene expression and PO activity, and induce the melanization.

Inhibitory effect of vascular endothelial growth factors-targeted small interfering RNA on proliferation of gastric cancer cells.

AIM: To examine the effects of vascular endothelial growth factor (VEGF)-targeted small interfering RNA (siRNA) on proliferation of gastric cancer cells in vitro. METHODS: Several siRNAs were transfected into human gastric cancer cell line SGC-7901 with Lipofectamine 2000. Cells not transfected with Lipofectamine 2000 or scrambled (SCR) siRNA served as controls. The inhibitory effect of siRNA on the expression of VEGF mRNA and protein was detected by RT-PCR and ELISA. MTT assay was used to examine the inhibition rate of cell growth. The change in cell cycling of siRNA-treated cells was detected by flow cytometry. RESULTS: siRNA targeting human VEGF effectively inhibited the proliferation of gastric cancer cell line SGC-7901 and the distribution of cell cycle. The percentage of G(0)/G(1) phase was significantly higher in siRNA(1)- and siRNA(2)-transfected cells than in control cells. The expression of VEGF mRNA was significantly inhibited in siRNA(1)- and siRNA(2)-transfected cells compared with that in control cells. VEGF protein notably decreased in siRNA-transfected cells, but had no effect on SCR siRNA. CONCLUSION: VEGF siRNA inhibits proliferation of gastric cancer cells in vitro.

[Clinical study on colonic transmission time and the effect of sini powder on it in functional constipation patients].

OBJECTIVE: To investigate the characteristic of colonic transmission in functional constipation (FC) and the effect of traditional Chinese medicine (TCM) Sini Powder (SP) on it. METHODS: The colonic transmission time (CTT) of 36 patients with FC (the FC group) and 22 healthy subjects (control group) was measured through colonic transmission test, and CTT of entire colon and that of various subsections was calculated with Hinton method and Arhan method respectively. After then, the FC group was treated with SP for 7 days, and CTT was detected again after treatment. RESULTS: Before treatment, body mass index (BMI) was higher, CTT of entire colon, left half colonic section, and sigmoid-rectal section were longer in the FC group than those in the control group (P < 0.05), no statistical difference in CTT of right half colon was found between the two groups (P > 0.05). After FC patients being treated with SP, their CTT of whole colon, left half colonic section and sigmoid-rectal section were significantly shortened (P < 0.05). CONCLUSION: FC patients were characterized by increased BMI and CTT prolonged and unevenly distributed in subsections, especially in the left half colon, sigmoid and rectum; SP could shorten the CTT in FC patients.

Two serine proteases from Anopheles dirus haemocytes exhibit changes in transcript abundance after infection of an incompatible rodent malaria parasite, Plasmodium yoelii.

Serine proteases are involved in regulation of innate immune responses, such as haemolymph coagulation, melanization reaction and antimicrobial peptide synthesis. Although several serine proteases have been characterized in Anopheles gambiae (A. gambiae), few were cloned from other malaria vectors. In this study, we identified three cDNA fragments of serine proteases (AdSp1, AdSp2 and AdSp3) from haemocytes of an oriental malaria vector, Anopheles dirus (A. dirus), by cloning of fragments amplified with degenerate primers into the T-vector. RT-PCR analysis demonstrated that both AdSp1 and AdSp3 genes were also expressed in salivary gland. Basic local alignment search tool (BLAST) search found that both AdSp1 and AdSp3 were highly similar in sequence to A. gambiae Sp14A and Sp14D2, insects prophenoloxidase activating enzyme (PPAE) and Drosophila protease easter. Semi-quantitative RT-PCR indicated the transcription level of both AdSp1 and AdSp3 in haemocytes of A. dirus infected with Plasmodium yoelii (P. yoelii) was significant higher than that fed on 5% glucose or normal mouse blood at 7 days after the infectious meal (p<0.05), when P. yoelii oocysts began to be melanized by A. dirus. Our results indicated that both AdSp1 and AdSp3 might play an important role during melanotic encapsulation of P. yoelii by A. dirus.

[cDNA cloning and location of Pagumogonimus skrj abini cysteine protease].

OBJECTIVE: To clone the cysteine protease cDNA fragment from Pagumogonimus skrjabini adults and locate the tissue of the adult worm where cysteine protease is expressed. METHODS: The cysteine protease cDNA fragment was amplified by reverse transcription-polymerase chain reaction (RT-PCR) with degenerated primers. The production was TA-cloned into the pUCm-T vector and sequenced. DNASIS program was used to analyse the nucleotide sequence and deduce the amino acid sequence, which was aligned with the correlated parasite cysteine protease afterwards. The digoxin labeled cRNA probe was synthesised by in vitro transcription with the cloned cDNA as template. The frozen sections of the adult worms were analysed by hybridization in situ to locate the gene expression. RESULTS: A 495 bp cDNA fragment was amplified by RT-PCR and sequenced. An amino acid sequence was deduced by DNASIS. Sequence analysis and alignment showed significant homologies with the correlated parasite cysteine proteinases and conservation of Cys, His and Asn residues that from a catalytic triad. In the hybridization in situ analysis, intestinal epithelium was stained positively on transverse section of adult worms. CONCLUSION: The cysteine proteinase cDNA fragment from Pagumogonimus skrjabini adults was cloned. There are some key sites which are correlated to the function of cysteine protease in the cDNA fragment. Cysteine protease is mainly expressed in intestinal epithelium of P. skrjabini.

[Study on the relationship between intracellular free calcium and melanization in oocysts of Plasmodium yoelii].

OBJECTIVE: To study the change of intracellular free Ca2+ in the oocyst when it melanized and to find out the relationship between the melanized oocyst and its intracellular level of free Ca2+ in a Plasmodium-refractory strain of Anopheles dirus. METHODS: The distribution and experimental condition of the intracellular free Ca2+ in oocyst of Plasmodium yoelii was measured with Ca2+ sensitive dye Fluo-3/AM and Pluronic F-127 under confocal laser scanning microscope (CLSM) at different time. RESULTS: The best load condition was that the oocysts were incubated in 3 mumol/ml Fluo-3/AM adding 1 microliter/ml 25% Pluronic F-127 for 60 min at 37 degrees C. Fluorescent imaging of oocysts was affected by an increase or decrease of the concentration of Fluo-3/Am and incubation time. The distribution of intracellular free Ca2+ was heterogeneous in the oocysts. The mean value of Ca2+ in the mature oocysts was (137.15 +/- 7.02) nmol/L (X +/- S) but was (18.44 +/- 1.75) nmol/L in melanized oocysts with Ca2+ sedimentation in the wall of oocyst. CONCLUSION: The results suggest that the level of the intracellular free Ca2+ in oocyst decreased and excreted during its melanization in a Plasmodium-refractory anopheline mosquito species.

[The internal control role of ribosomal protein S7 in the defense of Anopheles dirus against Plasmodium infection].

OBJECTIVE: To investigate the role of ribosomal protein S7 (rpS7) in the defense of Anopheles dirus against infection. METHODS: rpS7 was amplified from Anopheles dirus hemocytes with degenerated primers designed according to the conservative region of S7, rpS7 was then cloned using T/A cloning kit and the inserted fragment was sequenced. The difference of the transcript abundance of rpS7 from Anopheles dirus hemocyte among non-blood-fed (N), normal-blood-fed (B) and Plasmodium yoelii infected groups (I) was also analyzed by RT-PCR and gel scanning system at d1, d2, d3, d4, d7 and d11 after blood feeding. RESULTS: There is no significant difference of rpS7 signal between the three groups. CONCLUSION: Anopheles dirus S7 can be used as an internal control to study the role of Anopheles dirus related immune factors in Plasmodium infection.

[Assay of haemolymph protein concentration in Anopheles stephensi].

OBJECTIVE: To ascertain the changes of haemolymph protein concentration in adult Anopheles stephensi mosquitoes under different feeding conditions. METHODS: Haemolymph samples from four groups of adult An. stephensi, fed with sucrose solution, normal blood, plasmodium-infected blood and nitroquine, respectively, were collected by expulsion method. The concentration of haemolymph protein was examined by Bradford method. The results were analyzed automatically by Excel program. RESULTS: The level of protein concentration in the infected blood-fed group was higher than the sucrose solution group and normal blood group at day 8 after Plasmodium yoelii infection, the average concentration was 4.436, 3.080 and 3.092 micrograms/microliter, respectively. The haemolymph protein concentration (2.264 micrograms/microliter) in the nitroquine-administered mosquitoes was lower than the infected blood-fed mosquitoes. CONCLUSION: The haemolymph protein concentration of the adult An. stephensi decreases after the nitroquine administration, indicating that the haemolymph proteins may be involved in the melantotic encapsulation reaction of plasmodial oocysts.