Saudi Journal of Gastroenterology
Home About us Instructions Submission Subscribe Advertise Contact Login    Print this page  Email this page Small font sizeDefault font sizeIncrease font size 
Users Online: 5706 


 
Table of Contents   
ORIGINAL ARTICLE  
Year : 2016  |  Volume : 22  |  Issue : 1  |  Page : 30-36
MicroRNA-155-enhanced autophagy in human gastric epithelial cell in response to Helicobacter pylori


Department of Gastroenterology, 309 Hospital of Chinese Peoples Liberation Army, Beijing, China

Click here for correspondence address and email

Date of Submission02-Feb-2015
Date of Acceptance11-Apr-2015
Date of Web Publication12-Jan-2016
 

   Abstract 

Background/Aim: MicroRNAs (miRNAs) are a class of small noncoding RNAs acting as posttranscriptional gene expression regulators in many physiological and pathological conditions. MiR-155 is one kind of miRNAs that plays an important role in causing various diseases. However, the precise molecular mechanism of the ectopic expression of miR-155 in Helicobacter pylori infection remains poorly understood. Autophagy has recently been identified as an effective way to control the intracellular bacterium survival. In the present study, we demonstrate a novel role of miR-155 in regulating the autophagy-mediated anti-H. pylori response. Patients and Methods: Totally 86 H. pylori-positive patients together with 10 H. pylori-negative, healthy control subjects were included in the study. Correlation between immunohistochemical grades and miR-155 expression were determined. Molecular mechanism of miR-155 on regulation of autophagy and elimination of intracellular H. pylori were determined using the GES-1 cell model. Results: We found that overexpression of miR-155 by transfecting miR-155 mimics could significantly decrease the survival of intracellular H. pylori, and this process was through induction of autophagy. Furthermore, there was a significant correlation between miR-155 and immunohistochemical grades in H. pylori-positive patients, and miR-155 expression were decreased in the intestinal metaplasia group. Conclusions: The results have indicated that the miR-155 expression level plays a key role in immunity response against H. pylori and this might provide potential targets for the future treatment of H. pylori-related diseases.

Keywords: Autophagy, Helicobacter pylori, intestinal metaplasia, microRNA

How to cite this article:
Wu K, Zhu C, Yao Y, Wang X, Song J, Zhai J. MicroRNA-155-enhanced autophagy in human gastric epithelial cell in response to Helicobacter pylori. Saudi J Gastroenterol 2016;22:30-6

How to cite this URL:
Wu K, Zhu C, Yao Y, Wang X, Song J, Zhai J. MicroRNA-155-enhanced autophagy in human gastric epithelial cell in response to Helicobacter pylori. Saudi J Gastroenterol [serial online] 2016 [cited 2020 Aug 11];22:30-6. Available from: http://www.saudijgastro.com/text.asp?2016/22/1/30/173756


Helicobacter pylori is a gram-negative bacterium that plays an etiologic role in gastritis, peptic ulcer disease, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer. [1] Although H. pylori is generally considered as an extracellular microorganism, a growing number of evidence supports that at least a subset of H. pylori has an intraepithelial location and a minor fraction of H. pylori resides inside gastric epithelial cells. H. pylori is capable of surviving and persisting in host cells because of its repertoire of evading the host immune response. [2] Meanwhile, host cells also deploy a multitude of immune defense mechanisms to kill this pathogen. Scientists believe that a better understanding of the complex network within H. pylori-infected host surroundings might help to explore the diagnostic and treatment method.

Autophagy is an evolutionarily conserved process, which is involved in maintaining cytoplasmic homeostasis by degrading damaged organelles or misfolded proteins. [3] The autophagic cascade is initiated by the engulfment of cytoplasmic cargoes by an autophagosome, which then fuses with a late endosome to form the autolysosome, exposing the inner compartment to lysosomal hydrolases for degradation. [4] Recently, studies have suggested that autophagy may link the multistep process of intracellular H. pylori clearance. [5] Additionally, H. pylori can induce autophagy in gastric epithelial cells. [6] However, the mechanism by which H. pylori antagonize host autophagy remains to be elucidated.

As a class of small noncoding RNAs, miRNAs are highly conserved between different eukaryotic species and they function as key regulators of gene expression at the post-transcriptional level by targeting mRNAs for translational repression or degradation. Previous work has reported that microRNA-155 negatively regulated the release of proinflammatory cytokines and signal transduction during H. pylori infection. [7] In this study, we aim to describe our new discovery about the regulatory role of microRNA-155 in autophagy triggered by H. pylori infection in gastric epithelial cell.


   Patients and Methods Top


Antibodies and reagents

The GFP-LC3B plasmid was provided by Dr. Liwei Dong (International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai); the pcDNA-3.1-Rheb plasmid and pcDNA-3.1-3- RPS6KB2 plasmid were obtained from RiboBio Co., Ltd, Guangzhou; methyladenine (3-MA, M9281) and Rapamycin (Rapa, R8781) were purchased from Sigma; Antibodies against LC3B (L7543) were obtained from Sigma; antibodies for Rheb (4935), RPTOR (2280), RICTOR (2114), RPS6KB2 (2708), and MTOR (2972) were obtained from cell signaling.

Cell and bacteria culture

The nonmalignant gastric epithelial cell lines GES-1 were routinely cultured in RPMI 1640 medium (Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin in a humidified incubator containing 5% CO 2 at 37°C. The wild-type H. pylori strain 26695 was obtained from ATCC and grown as previously described. [8] Subsequently, cells were seeded to the wells of a 12-well plate and grown to 80% confluency. Then, the medium was replaced with antibiotic-free medium. H. pylori was added to cells at a multiplicity of infection of 100:1. The infection model was monitored by the release of interleukin-8 (IL-8) and growth-related oncogene-alpha (GRO-alpha), as measured by DuoSet ELISA Development System (R and D).

Cell transfection

All oligonucleotides were synthesized from GenePharma (Shanghai, P. R. China) products. Transfections were performed using Lipofectamine 2000 (Invitrogen). Cells were transfected with 50 nM miRNA mimics, inhibitors, or scrambled miR-control for 24 h.

Clinical samples

For clinical samples, a total number of 86 H. pylori-positive patients together with 10 H. pylori- negative but healthy control subjects were included in the study. The H. pylori infection status was confirmed by bacterial culture, C 13 -urea breath test, and the histologic testing. Patients were regarded as being H. pylori positive if one of the tests yielded positive results. Patients with a history of gastric surgery, active gastrointestinal bleeding, use of steroids, immunosuppressive drugs, NSAIDs, proton pump inhibitors, or who were treated for H. pylori eradication were excluded from the study. Gastric fragments were obtained during endoscopy from five different sites as recommended by the Updated Sydney System for classification of gastritis. [9] A number of clinicopathological variables such as gender, age, tumor location, histological type, tumor-node-metastasis (TNM) stage, depth of tumor invasion, lymph node metastasis, distant metastasis, and vascular invasion were obtained from the histopathological records and included for survival analysis. The tissue sections were also assessed for the presence of H. pylori infection by immunohistochemical staining using polyclonal anti-H. pylori antibody. Presence of H. pylori was graded according to the following criteria: Grade 0 (0 bacteria/oil immersion field), Grade 1 (19 bacteria/oil immersion field), Grade 2 (20-29 bacteria/oil immersion field), Grade 3 (30-99 bacteria/oil immersion field), and Grade 4 (≥100 bacteria/oil immersion field).

Quantitative reverse-transcription polymerase chain reaction

Total RNA was extracted from cells using the Trizol reagent (Invitrogen) in accordance with the manufacturer's protocol. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis for miRNAs was performed by using TaqMan miRNA assays (Ambion) in an iQ5 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Reverse transcription reactions were performed using the following parameters: 16°C, 30 min, 42°C, 30 min, and 84°C, 5 min. PCR reactions were performed using the following parameters: 95°C for 2 min followed by 40 cycles of 95°C, 15 s, and 60°C, 30 s. U6 small nuclear RNA was used as endogenous control for data normalization. Relative expression was calculated using the comparative threshold cycle (Ct) method. qRT-PCR analysis for the mRNAs of PTGS2 and β-actin was performed by using PrimeScript RT-PCR kits (Takara, Shiga, Japan). The mRNA level of β-actin was used as an internal control. The sequences of primers used were described in accordance with the previous report. [10]

Western blot

Cells were washed with ice-cold phosphate-buffered saline (PBS) and then lysed with protein lysate (Pierce, Rockford, IL, USA). After centrifugation at 5000 × g for 15 min at 4°C, the protein concentration was measured with a bicinchoninic acid (BCA) protein assay kit (Pierce). Fifty microgram aliquots of lysates were loaded on a sodium dodecylsulfate (SDS) polyacrylamide 10% gradient gel and transferred to a polyvinylidene difluoride membrane. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline, pH 7.4, containing 0.05% Tween 20, and were incubated with primary antibodies (1:200; Santa Cruz, CA, USA) and horseradish peroxidase-conjugated secondary antibodies (1:5000; Santa Cruz) in accordance with manufacturer's instructions. The protein of interest was visualized using an enhanced chemiluminescence (ECL) Western blotting substrate (Pierce) and the Chemidoc XRS Gel Documentation System (BioRad).

Statistical analyses

The results are expressed as means ± SD from at least three separate experiments performed in triplicate. The difference between groups was determined using two-tailed Student's t-test by using SPSS software (Armonk, NY, USA). P values of less than 0.05 were considered significant. The Chi-square test or Fisher's exact test was used to analyze the relationship between miR-155 expression and the clinicopathological features.


   Results Top


miR-155 decreases the survival of intracellular H. pylori

GES-1 cells were transiently transfected with miR-155 mimic or inhibitor, and then challenged with H. pylori at an MOI of 100. Intracellular survival of H. pylori in GES-1 cells was examined at different times. Our results showed that miR-155 significantly reduced the survival of intracellular H. pylori, whereas transfection with miR-155 inhibitor returned the opposite results [Figure 1]. These results indicated that miR-155 could enhance the host killing ability against intracellular H. pylori.
Figure 1: miR-155 decreases the survival of intracellular  Helicobacter pylori Scientific Name Search 1 cells. GES-1 cells were transiently transfected with negative control (NC), miR-155 mimic or miR-155 inhibitor, and then challenged with H. pylori at an MOI of 100, and intracellular H. pylori viability was determined by CFU counting at certain timepoint. MOI,: multiplicities of infection; CFU, colony forming units

Click here to view


miR-155 induces autophagy and promotes the elimination of intracellular H. pylori in GES-1 cell

Western-blot results showed that miR-155 enhanced the ratio of LC3-II/GAPDH, whereas miR-155 inhibitor reduced the LC3B-II/GAPDH ratio [Figure 2]a. To further confirm that miR-155 triggered the autophagy process, we used a GFP-LC3-II puncta formation assay to monitor autophagy. Transfection with miR-155 displayed a significant increase in the percentage of cells with autophagosomes (GFP-LC3-II dots) compared with mock-infected GES-1 cells (P < 0.05) [Figure 2]b. The collective data above demonstrated that miR-155 elevated the autophagic response in GES-1 cell.
Figure 2: miR-155 induces autophagy in GES-1 cells. (a) GES-1 cells were transfected with negative control (NC), miR-155 mimic or miR-155 inhibitor for 24 h followed by Helicobacter pylori infection (MOI = 100:1), and the ratio of LC3B-II/GAPDH were determined. (b) GES-1 cells were transfected with plasmid expressing GFP-LC3B, together with negative control (NC), miR-155 mimics or inhibitor. After 24 h, the cells were incubated for 6 h at 37°C in F12 medium with H. pylori. Following fixation, cells were immediately visualized by confocal microscopy. The number of GFP-LC3B puncta in each cell was counted. HP, H. pylori; MOI, multiplicities of infection; CFU, colony forming units

Click here to view


We next examined whether miR-155 decreased the survival of intracellular H. pylori by induction of autophagy. GES-1 cells were pretreated with miR-155 mimic or inhibitor, and either left untreated or pretreated with 3-methyladenine (3-MA) or Rapamycin, followed by exposing to H. pylori for 24 h. We have found that Rapamicyin helped miR-155 to reduce the survival of intracellular H. pylori, whereas 3-MA blocked this reduction [Figure 3]a. As expected, the opposite results were seen in the miR-155 inhibitor group [Figure 3]b. These data demonstrated that miR-155 decreased the survival of intracellular H. pylori by inducing the autophagy process.
Figure 3: miR-155 decreases the survival of intracellular Helicobacter pylori by induction of autophagy. Cells were pretreated with 3-MA, and then transfected with negative control (NC) and miR-155 mimics for 24 h. (a) The ratio of LC3B-II/GAPDH were determined by Western blotting (b) intracellular H. pylori viability was determined by CFU counting. MOI, multiplicities of infection; CFU, colony forming units

Click here to view


miR-155 induces autophagy by inhibiting Rheb

The previous report has demonstrated that miR-155 induced autophagy by targeting multiple players in the MTOR pathway. [10] Therefore, we then selected Rheb, RPTOR, RICTOR, RPS6KB2, and MTOR, which 3′- UTRs complementary to miR-155 were confirmed previously, to further examine the possible regulatory mechanism of microRNA-155-induced autophagy. First, we measured both the mRNA and the protein levels of these targets in GES-1 cells transfected with miR-155 mimics or inhibitors. The results showed that miR-155 mimics obviously decreased the expression of Rheb and RPS6KB2, whereas other target genes did not show significant changes [Figure 4]a and b. We overexpressed Rheb/RPS6KB2 by transient transfecting plasmids pcDNA3.1-Rheb/pcDNA3.1-RPS6KB2 for 24 h  following by H. pylori infection. As shown in [Figure 4]c, LC3B-II/GAPDH ratio was sharply reduced in the Rheb and RPS6KB2 overexpressed group, which indicated that Rheb and RPS6KB2 weakened the autophagy development. Cotransfection with microRNA-155 mimics could block this autophagy reduction in the Rheb overexpressed group, but not in the RPS6KB2 group. This suggested that microRNA-155 up-regulated autophagy by suppressing Rheb [Figure 4]c and d.
Figure 4: miR-155 induces autophagy by inhibiting Rheb. Cells were transfected with negative control (NC) and miR-155 mimics for 24 h, and (a) mRNA and (b) protein level of Rheb, RPTOR, RICTOR, RPS6KB2, and MTOR were determined. GES-1 cells were transfected with plasmid expressing (c) Rheb or (d) RPS6KB2, together with negative control (NC) or miR-155 mimics, and the ratio of LC3B-II/GAPDH were determined by western blotting

Click here to view


Correlation of miR-155 with clinical value

Then we analyzed the associations between miR-155 and their clinical values in human gastric mucosal tissues. Of the 86 H. pylori-positive, chronic gastritis patients, 77 (89.5%) H. pylori-infected patients displayed evidence of inflammatory infiltrates with lymphocytes and mononuclear cells, and 19 (22.1%) displayed intestinal metaplasia, whereas the H. pylori-negative subjects had mostly normal mucosa. miR-155 was upregulated in H. pylori-positive patients [[Figure 5]a, P < 0.05] and was positively correlated with the immunohistochemical grades [Figure 5]b. Furthermore, miR-155 expression was increased in the intestinal metaplasia group [Figure 5]c. We next followed up with eight patients who had showed significant release of H. pylori infection before and after the H. pylori treatment. Of these patients, six (75%) exhibited significant decreased value of miR-155 [Figure 5]c, which indicated that miR-155 could monitor the condition of H. pylori infection.
Figure 5: Correlation of miR-155 with clinical value in gastric mucosal tissues. (a) Differential miR-155 expression in gastric mucosal tissues. (b) Correlation of miR-155 and the immunohistochemical grades in gastric mucosal tissues among Helicobacter pylori positive patients. (c) Associations between miR-155 and intestinal metaplasia. IM: Intestinal metaplasia. (d) Comparing the differential miR-155 expression in gastric mucosal tissues in 8 patients before and after anti- H. pylori treatment

Click here to view



   Discussion Top


H. pylori may promote the development of inflammation of gastric mucosa, which then induces gastric atrophy and intestinal metaplasia, resulting in gastric cancer. This process is a comprehensive action associated with multiple factors, such as inhibition of tumor suppressor genes, overexpression of related genes and a failure to regulate cell proliferation. Studies have revealed that miR-155 participates in various biological pathogenesis of H. pylori-related diseases. Xiao et al. [7] have demonstrated that miR-155 might be involved in the function as a novel negative regulator to modulate the inflammation response in H. pylori infection. Oertli et al. [11] have indicated that miR-155 is essential for the T cell-mediated control of H. pylori infection. However, whether this microRNA plays a critical role in the regulation of survival of intracellular H. pylori remains unknown. Our present results demonstrate that miR-155 decreased the survival of intracellular H. pylori at all tested timepoints, which indicated that miR-155 promoted bacterial elimination. As we know, apoptosis is an important biological process for bacterial clearance, but some reports indicate that miR-155 may display a role in the apoptosis inhibition. Saito et al. [12] have demonstrated that miR-155 suppress the proapoptotic gene TP53INP1 as their target. Koch's study proposes that the antiapoptotic effects of miR-155 could enhance macrophage resistance to apoptosis induced by DNA damage during H. pylori infection. [13] So we hypothesize that there might be a different way for miR-155 to enhance the intracellular H. pylori killing.

Autophagy has been demonstrated as a new type of pathogenic bacteria killing mechanism. On testing the autophagy related genes LC3-II, we demonstrated that miR-155 could enhance H. pylori induced autophagy. It was shown that blocking autophagy by Rapamycin decreased the intracellular H. pylori count, which indicated that miR-155 promoted H. pylori clearance through upregulating autophagy. It has become increasingly recognized that altered autophagy is associated with decreasing H. pylori survival. Chu et al. [14] have reported that the autophagy inducer Rapamycin enhanced the clearance of the H. pylori, and H. pylori usurp the autophagic vesicles as the site for replication, and the autolysosomes after fusion also degraded the replicating bacteria. Tang et al. [6] have demonstrated that H. pylori infection increased MIR30B during in vivo and in vitro infections. Based on the present results, we propose that compromise of autophagy by miR-155 benefits the intracellular H. pylori from evading autophagic clearance, which may provide a better understanding of the host anti-H. pylori response.

Many genes have been experimentally validated in predicted targets of miR-155, some of which were closely involved in autophagy. Wan et al. [10] have reported that miR-155 was an autophagy inducer by targeting multiple players in the MTOR pathway. In this study, we elucidated that miR-155 downregulated the expression of Rheb and RPS6KB2, which were the key proteins in mTOR pathway, playing a key role in promoting autophagy to eliminate intracellular H. pylori. Tang et al. [15] have identified MyD88 as a novel target of miR-155, and MyD88 was demonstrated targeting Beclin 1 to trigger autophagy. [16] It has suggested that during its co-existence with humans, H. pylori, host, and environmental factors consist of a complex network to mediated autophagic processes.

Data of clinical samples showed a significant correlation between miR-155 and immunohistochemical grades in H. pylori-positive patients, which confirmed the in vitro results of our present work. The published data have shown that miR-155 play a potential role as diagnostic and prognostic indicators in various diseases including cancer. [17] When testing the relationship between miR-155 expression and diagnostic index, we found that the miR-155 expression was decreased in the intestinal metaplasia group. Considering that intestinal metaplasia was closely related with gastric cancer, we assumed that miR-155 expression would play a prognostic role in carcinogenesis of H. pylori. Our results indicated that miR-155 enhancement might eliminate intracellular H. pylori and therefore prevent the gastric cancer occurrence. Molecular mechanism study indicated that this process might through autophagy. In the future, microRNA-based treatments, in combination with traditional chemotherapy, may be a new strategy for the clinical management of H. pylori-related gastric cancer.

The main limitation of this work was that the study size is not big enough. Further studies on more patients are ongoing to effect the generalizability of our conclusion. In addition, the relationship between autophagy indicators and clinical index should also be determined.

 
   References Top

1.
Salama NR, Hartung ML, Müller A. Life in the human stomach: Persistence strategies of the bacterial pathogen Helicobacter pylori. Nat Rev Microbiol 2013;11:385-99.  Back to cited text no. 1
    
2.
Wang YH, Wu JJ, Lei HY. When Helicobacter pylori invades and replicates in the cells. Autophagy 2009;5:540-2.  Back to cited text no. 2
    
3.
Levine B, Kroemer G. Autophagy in the pathogenesis of disease. Cell 2008;132:27-42.  Back to cited text no. 3
    
4.
Puleston DJ, Simon AK. Autophagy in the immune system. Immunology 2014;141:1-8.  Back to cited text no. 4
    
5.
Yang JC, Chien CT. A new approach for the prevention and treatment of Helicobacter pylori infection via upregulation of autophagy and downregulation of apoptosis. Autophagy 2009;5:413-4.  Back to cited text no. 5
    
6.
Tang B, Li N, Gu J, Zhuang Y, Li Q, Wang HG, et al. Compromised autophagy by MIR30B benefits the intracellular survival of helicobacter pylori. Autophagy 2012;8:1045-57.  Back to cited text no. 6
    
7.
Xiao B, Liu Z, Li BS, Tang B, Li W, Guo G, et al. Induction of microRNA-155 during Helicobacter pylori infection and Its negative regulatory role in the inflammatory response. J Infect Dis 2009;200:916-25.  Back to cited text no. 7
    
8.
Wu K, Yang L, Li C, Zhu CH, Wang X, Yao Y, et al. MicroRNA-146a enhances Helicobacter pylori induced cell apoptosis in human gastric cancer epithelial cells. Asian Pac J Cancer Prev 2014;15:5583-6.  Back to cited text no. 8
    
9.
Shukla S, Pujani M, Agarwal A, Pujani M, Rohtagi A. Correlation of serology with morphological changes in gastric biopsy in Helicobacter pylori infection and evaluation of immunohistochemistry for H. pylori identification. Saudi J Gastroenterol 2012;18:369-74.  Back to cited text no. 9
[PUBMED]  Medknow Journal  
10.
Wan G, Xie W, Liu Z, Xu W, Lao Y, Huang N, et al. Hypoxia-induced MIR155 is a potent autophagy inducer by targeting multiple players in the MTOR pathway. Autophagy 2014;10:70-9.  Back to cited text no. 10
    
11.
Oertli M, Engler DB, Kohler E, Koch M, Meyer TF, Müller A. MicroRNA-155 is essential for the T cell-mediated control of Helicobacter pylori infection and for the induction of chronic Gastritis and Colitis. J Immunol 2011;187:3578-86.  Back to cited text no. 11
    
12.
Saito Y, Suzuki H, Tsugawa H, Imaeda H, Matsuzaki J, Hirata K, et al. Overexpression of miR-142-5p and miR-155 in gastric mucosa-associated lymphoid tissue (MALT) lymphoma resistant to Helicobacter pylori eradication. PLoS One 2012;7:e47396.  Back to cited text no. 12
    
13.
Koch M, Mollenkopf HJ, Klemm U, Meyer TF. Induction of microRNA-155 is TLR- and type IV secretion system-dependent in macrophages and inhibits DNA-damage induced apoptosis. Proc Natl Acad Sci U S A 2012;109:E1153-62.  Back to cited text no. 13
    
14.
Chu YT, Wang YH, Wu JJ, Lei HY. Invasion and multiplication of Helicobacter pylori in gastric epithelial cells and implications for antibiotic resistance. Infect Immun 2012;78:4157-65.  Back to cited text no. 14
    
15.
Tang B, Xiao B, Liu Z, Li N, Zhu ED, Li BS, et al. Identification of MyD88 as a novel target of miR-155, involved in negative regulation of Helicobacter pylori-induced inflammation. FEBS Lett 2010;584:1481-6.  Back to cited text no. 15
    
16.
Shi CS, Kehrl JH. MyD88 and Trif target Beclin 1 to trigger autophagy in macrophages. J Biol Chem 2008;283:33175-82.  Back to cited text no. 16
    
17.
Xu TP, Zhu CH, Zhang J, Xia R, Wu FL, Han L, et al. MicroRNA-155 expression has prognostic value in patients with non-small cell lung cancer and digestive system carcinomas. Asian Pac J Cancer Prev 2013;14:7085-90.  Back to cited text no. 17
    

Top
Correspondence Address:
Dr. Kai Wu
Department of Gastroenterology, 309 Hospital of Chinese Peoples Liberation Army, Beijing - 100 091
China
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1319-3767.173756

Rights and Permissions


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]



 

Top
  
 
  Search
 
  
  
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Email Alert *
    Add to My List *
* Registration required (free)  


    Abstract
   Patients and Methods
   Results
   Discussion
    References
    Article Figures

 Article Access Statistics
    Viewed2457    
    Printed29    
    Emailed0    
    PDF Downloaded593    
    Comments [Add]    

Recommend this journal