[Study on mechanisms of Th17/Treg imbalance in patients with cystic echinococcosis based on miRNA expression profiles].

OBJECTIVE: To investigate the serum microRNA (miRNA) expression and examine the impact of miRNA expression profiles on T helper type 17 (Th17)/regulatory T cells (Treg) imbalance among patients with cystic echinococcosis, so as to provide insights into the illustration of the mechanisms underlying chronic Echinococcus granulosus infections, and long-term pathogenesis. METHODS: Total RNA was extracted from the sera of cystic echinococcosis patients and healthy controls, and subjected to high-throughput sequencing with the Illumina sequencing platform. Known miRNAs were annotated and new miRNAs were predicted using the miRBase database and the miRDeep2 tool, and differentially expressed miRNAs were identified. The target genes of differentially expressed miRNAs were predicted using the software miRanda and TargetScan, and the intersection was selected for Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Among the differentially expressed miRNAs with the 20 highest fold changes, miRNAs that targeted genes relating to key transcription factors RORC and FOXP3 that determine the production of Th17 and Treg cells or their important regulatory pathways (PI3K-Akt and mTOR pathways) were matched. RESULTS: A total of 53 differentially expressed miRNAs were screened in sera of cystic echinococcosis patients and healthy controls, including 47 up-regulated miRNAs and 6 down-regulated miRNAs. GO enrichment analysis showed that these differentially expressed miRNA were involved DNA transcription and translation, cell components, cell morphology, neurodevelopment and metabolic decomposition, and KEGG pathway analysis showed that the differentially expressed miRNA were mainly involved in MAPK, PI3K-Akt and mTOR signaling pathways. Among the differentially expressed miRNAs with the 20 highest fold changes, there were 3 miRNAs that had a potential for target regulation of RORC, and 15 miRNAs that had a potential to target the PI3K-Akt and mTOR signaling pathways. CONCLUSIONS: Significant changes are found in serum miRNA expression profiles among patients with E. granulosus infections, and differentially expressed miRNAs may lead to Th17/Treg imbalance through targeting the key transcription factors of Th17/Treg or PI3K-Akt and mTOR pathways, which facilitates the long-term parasitism of E. granulosus in hosts and causes a chronic disease.

[The application of driver stress inventory in Chinese drivers and its reliability and validity test].

To explore and revise the factor structure, reliability and validity of the Chinese version of the Driver Stress Inventory (DSI) in the driver population in first-tier city of China. In this study, the questionnaire method was used to select the data collected by the "Research on the Driving Stress of Urban Salaries in Urban Traffic" carried out by the Institute of Psychology, Chinese Academy of Sciences in 2012, and a third-party survey agency was commissioned to select 300 people in Beijing, Shanghai, and Guangzhou respectively. A total of 900 drivers (with 889 valid data scales) were used to test their driving stress and other indicators by using the DSI and the self-compiled travel and stress relief scale. The test content collects driver self-reported stress data from five dimensions: Aggression, Dislike of driving, Hazard-Monitoring, Fatigue Proneness and Thrill-seeking. The DSI questionnaire of 445 cardinal numbers was revised by item analysis methods such as correlation test and T-test, and exploratory factor analysis method based on principal component analysis and optimal skew axis method (Promax).The results of item analysis showed that the total correlation coefficient of 10 items of the 48 items of the original DSI scale was lower than 0.3, and the total correlation coefficient of 6 items was not significant (r=-0.078-0.079, P>0.05), and the high and low groups were independent. There were significant differences in the results of the sample t test (t=-16.642-0.091, P<0.001), the 16 items were deleted, and the remaining 32 items; exploratory factor analysis showed that KMO=0.938>0.900, and the Bartlett's sphericity test result was significant (χ²=6 361.974, df=496, P<0.001), suitable for exploratory factor analysis, the results showed that 2 items constituted independent factors, did not meet the relevant standards of psychometrics and were deleted, and finally retained 30 items, and the internal consistency coefficient of the new scale was better than the original one(α=0.932>0.877); Based on the results of exploratory factor analysis, the model fitting indexes of 444 even-numbered samples such as RMSEA, SRMR, CFI and TLI were verified by confirmatory factor analysis, and the results showed that the index of each index was good (χ²=1 250.447, RMSEA=0.070, SRMR=0.068, CFI=0.839, TLI=0.823); criterion validity analysis found that each factor of the revised scale was significantly correlated with situational anxiety (r=0.190-0.556, P<0.01). In conclusion, the DSI (Chinese version) has good reliability and validity, and can be used as an assessment tool for driver stress in China.

Lamin B1 deficiency promotes malignancy and predicts poor prognosis in gastric cancer.

Gastric cancer (GC) is a kind of global malignancy. However, the expression pattern and clinical relevance of lamin B1 in GC remain to be elucidated. We endeavored to investigate how GC is influenced by lamin B1 and the related mechanisms. The lamin B1 expression in GC tissues from 71 patients was assessed by using immunohistochemistry (IHC). The expression of lamin B1 was connected with the clinical stage, depth of invasion, and poorer overall survival. Colony formation assays and methyl thiazolyl tetrazolium (MTT) were used to assess cell viability. The migration ability of GC cells was determined by cell scratch assay and Transwell invasion assay. Moreover, we used two cell lines of GC to explore the underlying mechanism of lamin B1 in boosting the GC cells proliferation and invasion in vitro by assessing the effects on related signal transduction pathways. Our data demonstrated that the expression level of lamin B1 was downregulated in GC tissues, and low expression level of lamin B1 was significantly correlated with higher clinical stage, depth of invasion, nodal stage, and poor prognosis. Moreover, in vitro experiments demonstrated that lamin B1 knockdown promoted, whereas lamin B1 overexpression inhibited, gastric cancer cell proliferation and migration. We also observed that lamin B1 knockdown could promote the activity of the PI3K/PTEN/Akt and MAPK/ERK pathway with a decrease in the p53/p21WAF1/CIP1 expression, whereas lamin B1 overexpression contributed to the opposite results. In conclusion, our studies indicate that lamin B1 deficiency is crucial in GC progression. Furthermore, the results elucidating the biological mechanisms of lamin B1 may potentially contribute to current GC treatment modalities.

Identification and diversity of Y-chromosome haplotypes in Qinghai yak populations.

The aim of the present study was to perform a preliminary analysis of the characterization and diversity of Y-chromosome haplotypes/haplogroups in yak of Qinghai Province, China. A total of 322 male yaks from nine populations belonging to three officially recognized breeds (Gaoyuan, Huanhu and Datong) were sampled. Animals were genotyped using six previously reported Y-SNPs present in the SRY, USP9Y, UTY, AMELY and OFD1Y genes and four new Y-SNPs in the OFD1Y gene (g.569A>C, g.578A>C, g.608G>T and g.653G>C) identified in this study. Seven Y-chromosome haplotypes (H1-H7) were identified according to the combination of the 10 Y-SNPs. H1, H2 and H6 were the most common and shared haplotypes across all yak populations/breeds. Private haplotypes H3 and H7 were detected in the Datong breed; H4 in Guoleimude, Qumalai, Qilian, Tianjun and Ganglong populations; and H5 in Qumalai of Gaoyuan breed. Haplotype clustering and network analyses inferred two haplogroups, Y1 and Y2, indicating two divergent lineages of paternal origins of Qinghai yak. The analysis of molecular variance showed a significant difference among individuals (P < 0.0001) with more than 93% of the total genetic variation present within populations, suggesting a weak genetic structure among Qinghai yak populations. The overall Y-haplotype diversity was 0.538 ± 0.028, showing a relatively high diversity in Qinghai yak. The Gaoyuan and Datong breeds had similar haplotype diversities (0.547 ± 0.030 and 0.553 ± 0.083, respectively), which were higher than that of the Huanhu breed (0.441 ± 0.098). Our results support the conservation and sustainable use of unique yak genetic resources in Qinghai.

Highly Conserved Keratin-Associated Protein 7-1 Gene in Yak, Taurine and Zebu Cattle.

Keratin-associated proteins (KRTAPs) play a critical role in cross-linking the keratin intermediate filaments to build a hair shaft. The genetic polymorphisms of the bovine KRTAP7-1 gene were investigated for the first time in this study. The complete coding sequence of the KRTAP7-1 gene in 108 domestic yak, taurine and zebu cattle from China and Indonesia were successfully amplified using polymerase chain reaction and then directly sequenced. Only two single-nucleotide polymorphisms (one nonsynonymous at c.7C/G and another synonymous at c.21C/T) and three haplotypes (BOVIN-KRTAP7-1*A, B and C) were identified in the complete coding sequence of the bovine KRTAP7-1 gene among all animals. There was no polymorphism across three Chinese indigenous yak breeds and one Indonesian zebu cattle population, all sharing the BOVINKRTAP71*A haplotype. The four taurine cattle populations also had BOVIN-KRTAP7-1*A as the most common haplotype with a frequency of 0.80. The frequency of novel haplotype BOVIN-KRTAP7-1*B was only 0.07 present in one heterozygous animal in each of the four taurine cattle populations, while BOVINKRTAP7- 1*C was only found in a Simmental and a local Chinese Yellow cattle population with frequencies of 0.17 and 0.36, respectively. The monomorphic yak KRTAP7-1 gene in particular, and highly conserved bovine, sheep and goat KRTAP7-1 genes in general, demonstrated its unique intrinsic structural property (e.g., > 21% high glycine content) and primary functional importance in supporting the mechanical strength and shape of hair.

[Prevention for glucocorticoid-induced osteonecrosis of femoral head: a long-term clinical follow-up trail].

Objective: To evaluated the outcome of prevention and treatment for glucocorticoid-induced osteonecrosis of femoral head with anticoagulant and vasodilator drugs. Methods: A prospective, randomized, double-blind study was performed. From August 2003 to August 2006, 58 patients with large amounts of hormone therapy in the Zhongshan Hospital Affiliated Dalian University were enrolled and randomly assigned to the control group (placebo) or preventive group (anticoagulant and vasodilator drugs). And we prospectively analyzed the clinical outcomes of 24 patients with glucocorticoid-induced osteonecrosis of femoral head early stage (treatment group)treated by anticoagulant and vasodilator drugsat the same time. Disease incidence rate and progression were evaluated by radiography and magnetic resonance imaging (MRI), Follow-up of patients with femoral head survival curve was drawn. The Harris Hip Score and the Short Form 36 health survey were used to rate hip function and quality of life, respectively. Results: Thus, a total of 80 patients were assessed in this study, 24 cases in control group[follow up from 7.5 to 13.0(10.7±1.6)years], 22 cases in preventive group and 24 cases in treatment group. There was significant difference in theincidence rate of Osteonecrosis of femoral head, survive rate of femoral head and HHS score between the control groupand preventive group(41.7% vs 13.6%, 66.7% vs 70.8% , P<0.01). Conclusion: Anticoagulant and vasodilator drugs could effect on preventing theglucocorticoid-induced osteonecrosis of femoral head, reducing disease progression, or improving life quality.

Serological investigation of Mycoplasma synoviae infection in China from 2010 to 2015.

Mycoplasma synoviae can cause worldwide respiratory diseases, synovitis, or subclinical symptoms in poultry. M. synoviae infection occurs throughout the yr and causes significant economic losses, including increased mortality, condemnations, medication, and live production cost. In the present study, the seroprevalence of M. synoviae among 44,395 non-vaccinated chickens from 21 provinces in China from 2010 to 2015 was estimated using ELISA. The overall seroprevalence was 41.19%. Seropositive rates in different yr ranged between 24.70 and 57.20%; the highest seropositive rate was observed in 2010, and the lowest was observed in 2013. The prevalence rates varied greatly in different provinces from 5.10 to 100%. Of the 463 commercial flocks tested, 375 (80.99%) were positive for M. synoviae by ELISA. The seasonal distribution ranged between 26.83% (in October) and 53.98% (in July). An investigation of chickens according to age further showed that M. synoviae can infect chickens at any age. Our findings indicate that M. synoviae infection is very common in China and should prompt further research into its prevalence to develop effective control and prevention strategies.

Nogo-A antibody treatment enhances neuron recovery after sciatic nerve transection in rats.

OBJECTIVE: The use of an antibody to block the neurite outgrowth inhibitor Nogo-A has been of great interest for promoting axonal recovery as a treatment for peripheral nerve injuries. The present study aimed to investigate the signaling pathway of p75 neurotrophin receptor (NTR) and Nogo receptor (NgR) in a sciatic nerve transection (SNT) rat model and evaluate the underlining mechanisms. MATERIALS AND METHODS: Seventy-five Sprague-Dawley (SD) rats were randomly divided into 3 groups (n=25), namely the sham group, sciatic nerve transection (model) group and Nogo-A-pAb group. Following euthanasia, spinal cord and sciatic nerve of the operation site were harvested, fixed in formalin. Hematoxylin and eosin (HE) staining was used to evaluate the sciatic nerve pathology. The mRNA and protein expression levels of Nogo-A, NTR were assessed by Real-time polymerase chain reaction (RT-PCR) and Western blotting, respectively. RESULTS: Histology showed enhanced regeneration of spinal axon in the anti-Nogo-A antibody group. At 48 hours after operation, mRNA of Nogo-A and NTR were higher in model group compared with control group. mRNAs were at their highest levels at 1 week, while these were at normal levels after 4 weeks in Nogo-A-pAb group. The protein levels of Nogo-A and NTR were higher in model group compared with sham-operation group at 1-week after operation; Nogo-A-pAb could reduce these proteins levels. CONCLUSIONS: The results suggested Nogo-A antibody might represent a promising repair strategy to promote recovery following SNT.

Renal clear cell carcinoma: diffusion tensor imaging diagnostic accuracy and correlations with clinical and histopathological factors.

AIM: To investigate whether diffusion tensor imaging (DTI) can be used to assess renal clinical histopathology, including the nuclear grade (NG), cell density (CD), and the presence of ki-67. MATERIALS AND METHODS: Thirty patients were enrolled in the study and were confirmed at surgical histopathology to have clear cell renal cell carcinoma (CCRCC). For DTI, a coronal echo-planar imaging sequence was performed (1400 ms repetition time, 76 ms echo time, diffusion direction=6, number of excitations=4; b=0 and 800 s/mm2, 6 mm section thickness with no intersection gap). CD and the presence of ki-67 were compared between the different NGs. Correlations between apparent diffusion coefficients (ADCs), E1, fractional anisotropy (FA), CD, and ki-67 were evaluated. RESULTS: ADC, E1, and FA values are important tools used to identify NG. The cut-off values were 1.003×10-3 mm2/s, 1.277×10-3 mm2/s, and 0.218 mm2/s, respectively. The difference between high- and low-grade CD was significant (t=-4.50, p<0.05). Similarly, a significant difference between high and low grade was also found in ki-67 (t=-4.03, p<0.05). ADC, E1, and FA values were decreased with increased CD; a significant negative correlation was found (r=-0.796, -0.865, and -0.996, respectively). Significant negative correlations between ADC, E1, and FA values, and ki-67 were found (r=-0.739, -0.826, and -0.876, respectively). CONCLUSIONS: DTI can be used to non-invasively assess CCRCC.

[Effects and underlying mechanism of berberine on renal tubulointerstitial injury in diabetic rats].

Objective: To investigate the effect of Berberine on renal tubulointerstitial injury and its potential mechanism in rats with type 2 diabetes mellitus (T2DM). Methods: Thirty Sprague-Dawley rats were randomly divided into 3 groups: normal control rats (NC group), diabetic rats without drug treatment (DM group), diabetic rats treated with Berberine (BBR group) for 8 weeks. At the end of the study, blood and urine samples were collected for biochemical examination, and tubulointerstitial fibrosis was quantified by Hematoxylin and Eosin (HE) and Masson staining. The expressions of E-cadherin (E-cad), α-smooth muscle actin (α-SMA), nuclear factor-κB (NF-κB) and monocyte chemoattractant protein 1 (MCP-1) were detected by immunohistochemistry analysis, real-time polymerase chain reaction (RT-PCR) and Western blot analysis. Results: 24 h urinary microalbumin (mAlb)[(170.5±58.1) vs (253.7±53.0) mg]and urinary N-acetyl-glucosaminidase (NAG)[(33.5±7.2) vs (49.5±9.3)U/L]in diabetic rats were significantly decreased by BBR treatment(both P<0.05). The apparent renal tubulointerstitial injury was found in the DM group, which was ameliorated by BBR treatment. The expression of α-SMA, NF-κB and MCP-1 were significantly decreased, accompanied by increased expression of E-cad in BBR-treated DM rats (all P<0.05). Conclusion: BBR could ameliorate renal tubulointerstitial injury in diabetic rats, the mechanism of which may be associated with the amelioration of epithelial-mesenchymal transition (EMT) through suppressing the expression of the NF-κB and MCP-1.

Genetic diversity and classification of Tibetan yak populations based on the mtDNA COIII gene.

To determine the level of genetic diversity and phylogenetic relationships among Tibetan yak populations, the mitochondrial DNA cytochrome c oxidase subunit 3 (COIII) genes of 378 yak individuals from 16 populations were analyzed in this study. The results showed that the length of cytochrome c oxidase subunit 3 gene sequences was 781 bp, with nucleotide frequencies of 29.2, 29.4, 26.1, and 15.2% for T, C, A, and G, respectively. A total of 26 haplotypes were identified, with 69 polymorphic sites, including 11 parsimony-informative sites and 58 single-nucleotide polymorphism sites. No deletions/insertions were found in sequence comparison, indicating that nucleotide mutation types were transitions and transversions. Haplotype and nucleotide diversities were 0.562 and 0.00138, respectively, indicating a high level of genetic diversity in Tibetan yak populations. Phylogenetic relationship analysis indicated that Tibetan yak populations are divided into 2 groups.

Y-STR INRA189 polymorphisms in Chinese yak breeds.

To further explore Y-STR INRA189 polymorphisms in the yak, and to determine the genetic differences among yak breeds, genotyping analysis of INRA189 in 102 male yak individuals from three yak breeds in Qinghai Province of China was performed. Genotyping revealed the presence of four alleles, with sizes of 149, 155, 157, and 159 bp, respectively. Of these, the 157-bp allele, which was found with the highest frequency in the three yak breeds, was the dominant allele. Interestingly, the 149-bp allele was only detected in the Gaoyuan breed, and the 159-bp allele was only found in the Huanhu and Datong breeds. Only the 157- and 155-bp alleles were found in all three yak breeds. Taking the three yak breeds as a single population, the frequency of these four alleles was 0.0294, 0.0686, 0.8628, and 0.0392, respectively. The average polymorphism information content in the three yak breeds was 0.2379, indicating that the INRA189 was a low polymorphic Y-STR marker in yak.

Novel Y-chromosome polymorphisms in Chinese domestic yak.

Y-chromosome-specific haplotypes (Y-haplotypes) constructed using single nucleotide polymorphisms (Y-SNPs) in the MSY (male-specific region of the Y-chromosome) are valuable in population genetic studies. But sequence variants in the yak MSY region have been poorly characterized so far. In this study, we screened a total of 16 Y-chromosome-specific gene segments from the ZFY, SRY, UTY, USP9Y, AMELY and OFD1Y genes to identify Y-SNPs in domestic yaks. Six novel Y-SNPs distributed in the USP9Y (g.223C>T), UTY19 (g.158A>C and g.169C>T), AMELY2 (g.261C>T), OFD1Y9 (g.165A>G) and SRY4 (g.104G>A) loci, which can define three Y-haplotypes (YH1, YH2 and YH3) in yaks, were discovered. YH1 was the dominant and presumably most ancient haplotype based on the comparison of UTY19 locus with other bovid species. Interestingly, we found informative UTY19 markers (g.158A>C and g.169C>T) that can effectively distinguish the three yak Y-haplotypes. The nucleotide diversity was 1.7 × 10(-4) ± 0.3 × 10(-4) , indicating rich Y-chromosome diversity in yaks. We identified two highly divergent lineages (YH1 and YH2 vs. YH3) that share similar frequencies (YH1 + YH2: 0.82-0.89, YH3: 0.11-0.18) among all three populations. In agreement with previous mtDNA studies, we supported the hypothesis that the two highly divergent lineages (YH1 and YH2 vs. YH3) derived from a single gene pool, which can be explained by the reunion of at least two paternal populations with the divergent lineages already accumulated before domestication. We estimated a divergence time of 408 110 years between the two divergent lineages, which is consistent with the data from mitochondrial DNA in yaks.

Technical note: Selection of suitable reference genes for studying gene expression in milk somatic cell of yak (Bos grunniens) during the lactation cycle.

Quantitative real-time PCR is the most sensitive technique for gene expression analysis. Data normalization is essential to correct for potential errors incurred in all steps from RNA isolation to PCR amplification. The commonly accepted approach for normalization is the use of reference gene. Until now, no suitable reference genes have been available for data normalization of gene expression in milk somatic cells of lactating yaks across lactation. In the present study, we evaluated the transcriptional stability of 10 candidate reference genes in milk somatic cells of lactating yak, including ACTB, B2M, GAPDH, GTP, MRPL39, PPP1R11, RPS9, RPS15, UXT, and RN18S1. Four genes, RPS9, PPP1R11, UXT, and MRPL39, were identified as being the most stable genes in milk somatic cells of lactating yak. Using the combination of RPS9, PPP1R11, UXT, and MRPL39 as reference genes, we further assessed the relative expression of 4 genes of interest in milk somatic cells of yak across lactation, including ELF5, ABCG2, SREBF2, and DGAT1. Compared with expression in colostrum, the overall transcription levels of ELF5, ABCG2, and SREBF2 in milk were found to be significantly upregulated in early, peak, and late lactation, and significantly downregulated thereafter, before the dry period. A similar pattern was observed in the relative expression of DGAT1, but no significant difference was revealed in its expression in milk from late lactation compared with colostrum. Based on these results, we suggest that the geometric mean of RPS9, PPP1R11, UXT, and MRPL39 can be used for normalization of real-time PCR data in milk somatic cells of lactating yak, if similar experiments are performed.

Association between the c.910A>G genetic variant of the XRCC1 gene and susceptibility to esophageal cancer in the Chinese Han population.

Esophageal cancer (EC) is a common malignancy worldwide. The X-ray repair cross-complementing 1 gene (XRCC1) is one of the most important candidate genes for influencing susceptibility to EC. This study aimed to investigate the effect of XRCC1 genetic variants on susceptibility to EC. A total of 383 EC patients (males: 239, females: 144, mean age: 56.62) and 387 cancer-free controls (males: 251, females: 136, mean age: 58.23) were enrolled in this study. The c.910A>G genetic variant of the XRCC1 gene was determined by polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing methods. The allele and genotype frequencies indicated statistical differences between EC patients and cancer-free controls. The c.910A>G genetic variant was statistically associated with increased susceptibility to EC [GG vs AA: odds ratio (OR)=1.79, 95% confidence interval (CI)=1.12-2.86, P=0.014; GG vs AG/AA: OR=1.76, 95%CI=1.13-2.75, P=0.013; G vs A: OR=1.25, 95%CI=1.01-1.55, P=0.041]. The allele G and genotype GG could contribute to the increased susceptibility to EC. Our findings suggest that the c.910A>G genetic variant is associated with susceptibility to EC in the Chinese Han population, and might be used as a molecular marker for detecting susceptibility to EC.

Association between the c.910A>G genetic variant of the XRCC1 gene and susceptibility to esophageal cancer in the Chinese Han population

Esophageal cancer (EC) is a common malignancy worldwide. The X-ray repair cross-complementing 1 gene (XRCC1) is one of the most important candidate genes for influencing susceptibility to EC. This study aimed to investigate the effect of XRCC1 genetic variants on susceptibility to EC. A total of 383 EC patients (males: 239, females: 144, mean age: 56.62) and 387 cancer-free controls (males: 251, females: 136, mean age: 58.23) were enrolled in this study. The c.910A>G genetic variant of the XRCC1 gene was determined by polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing methods. The allele and genotype frequencies indicated statistical differences between EC patients and cancer-free controls. The c.910A>G genetic variant was statistically associated with increased susceptibility to EC [GG vs AA: odds ratio (OR)=1.79, 95% confidence interval (CI)=1.12-2.86, P=0.014; GG vs AG/AA: OR=1.76, 95%CI=1.13-2.75, P=0.013; G vs A: OR=1.25, 95%CI=1.01-1.55, P=0.041]. The allele G and genotype GG could contribute to the increased susceptibility to EC. Our findings suggest that the c.910A>G genetic variant is associated with susceptibility to EC in the Chinese Han population, and might be used as a molecular marker for detecting susceptibility to EC.

Technical note: identification of suitable normalizers for microRNA expression analysis in milk somatic cells of the yak (Bos grunniens).

MicroRNA are approximately 18- to 22-nucleotide nonprotein coding molecules that play important roles in the regulation of gene expression at the posttranscriptional level. In the present study, we assessed the suitability of 8 noncoding small RNA as normalizers for microRNA (miR) quantitative analysis in milk somatic cells of lactating yaks, including 3 small nuclear RNA (snRNA; RNU1A, RNU5A, and RNU6B), 3 small nucleolar RNA (snoRNA; SNORA73A, Z30, and SNORA74A), 1 rRNA (5S), and 1 transfer RNA (Met-tRNA). The snRNA RNU1A, RNU5A, and SNORA73A were identified as the most stable references in milk somatic cells of lactating yaks. Also, a minimum of 3 reference RNA (RNU1A, RNU5A, and SNORA73A) were required for the normalization of microRNA expression data in milk somatic cells of the lactating yak. We further evaluated the suitability of the combination of RNU1A, RNU5A, and SNORA73A as reference RNA in milk somatic cells of lactating yaks via detecting the relative expression of miR 16b, miR 21-5p, miR 145, and miR 155 as microRNA of putative interest. In comparison to the colostrum period, on the whole, the expressions of the 4 microRNA were found to be upregulated at an early period and, thereafter, a declining pattern was exhibited from early to final periods in all microRNA investigated. Based on the results from this study, we recommend that the combination of RNU1A, RNU5A, and SNORA73A can be used as normalizers for microRNA quantitative analysis in future longitudinal studies on milk somatic cells of lactating yaks in relation to lactation.