The hepatocyte-specific Lass2-KO mice used in the present study were generated by the crossing of mice (C57BL/6J) carrying floxed the second exon of Lass2 and Albumin-Cre transgenic mice (C57BL/6J), as previously reported (5). All protocols for animal care and use were approved by the Regulations for the Administration of Affairs Concerning Experimental Animals (The State Science and Technology Commission of P.R. China, 1988), and the Animal Experimental Center of Jiangsu University was licensed for animal experiments. All of the mice were housed in pathogen-free (SPF) animal facilities under a standard 12-h-light/12-h-dark cycle. Animals received free access to water and commercial mouse chow throughout the present study. One-month-old male Lass2-KO and -WT mice were sacrificed by cervical dislocation. The mice for the experiments that followed were n=10/group. Liver weight/body weight were measured.

The mice were sacrificed by cervical dislocation, and blood harvested from the inferior vena cava was centrifuged at 1,600 × g for 10 min at room temperature to obtain serum for hepatic biochemical estimation. The activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) in serum were estimated using an AutoAnalyzer (Hitachi 7600, Japan) at the Affiliated Hospital of Jiangsu University.

Liver tissues were immediately removed from the sacrificed mice, partly fixed in AAF (100% alcohol 85 ml, acetic acid 5 ml, formalin 10 ml) for morphological examination, and partly stored at −80°C for further use. The tissues were paraffin-embedded and sectioned (5-μm thick). Periodic acid-Schiff (PAS) staining was performed using a commercially available kit (cat. no. 0609A14; Shellfish Gamma Biotechnology Co., Ltd., Nanjing, China). Briefly, the sections were incubated in 0.5% periodic acid solution for 15 min, rinsed with distilled water, and exposed to Schiff’s reagent for 20 min followed by two 3-min exposures to 0.6% sodium metabisulfite.

Tissues were lysed in RIPA lysis buffer containing the protease inhibitor phenylmethanesulfonyl fluoride (cat. nos. P0013C and ST506; both from Beyotime Institute of Biotechnology). The cell extracts were centrifuged at 12,000 × g for 20 min at 4°C in a Beckman Avanti-30 centrifuge, and the supernatants were used for the experiments. The protein concentrations were determined with the BCA assay kit (cat. no. P0009; Beyotime Institute of Biotechnology). The equivalent tissue proteins (10 μg/lane) were subjected to electrophoresis on a Mini-Protean Tetra Electrophoresis System (165–8001; Bio-Rad, Hercules, CA, USA) and transferred onto PVDF membranes (Millipore, Bedford, MA, USA) via a semi-dry transfer system (Bio-Rad). The membranes were blocked with 5% non-fat milk for 1 h at room temperature in TBST [50 mM (pH 7.5) Tris, 0.9% NaCl and 0.1% Tween-20] and then incubated with a 1:200 dilution of rabbit anti-mouse/human albumin (cat. no. BS6520; Bioworld Technology, Inc., Dublin, OH, USA) overnight at 4°C. The membranes were washed and then incubated with peroxidase goat anti-rabbit antibody (cat. no. XR-9920; ProSci Inc., Poway, CA USA) for 1 h at room temperature and developed using the BeyoEcl Plus kit (cat. no. P0018; Beyotime Institute of Biotechnology) for 1 min, and then scanned by ChampChemi professional (SG2011; Beijing Sage Creation Science Co., Ltd., Beijing, China).

Total RNA including miRNA from liver tissues was extracted by TRIzol (cat. no. 15596026; Invitrogen Corp., Carlsbad, CA, USA) or the miRNeasy Mini kit (cat. no. 217004; Qiagen, Hilden, Germany) according to the manufacturer’s suggestions. For mRNA qPCR, RNA was transcribed into cDNA using the QuantiTect reverse transcription kit and QuantiTect SYBR-Green PCR kits (cat. no. 205311 and no. 204243; both from Qiagen) according to the manufacturer’s protocols. For miRNA qPCR, RNA was transcribed into cDNA using the miScript Reverse II transcription kit (cat. no. 218161; Qiagen). The reaction component consisted of total RNA 1 μg, miScript HiSpec buffer 4 μl, Nucleics Mix 2 μl, miScript reverse transcriptase mix 2 μl, RNase-free H₂O up to 20 μl. The reaction was carried out at 37°C for 60 min and 95°C for 5 min on the ABI PCR 9700 system (Applied Biosystems, Foster City, CA, USA). cDNA was diluted in 80 μl nuclease-free H₂O for further application by LightCycler 480 SYBR-Green I master (Roche, Switzerland; cat. no. 04887352001). The reaction system for qPCR consisted of: LightCycler 480 SYBR-Green I Master 5 μl, forward primer 0.2 μl, reverse primer 0.2 μl, cDNA 1 μl, nuclease-free H₂O 3.6 μl. PCR was run on ABI 7500 Fast (Applied Biosystems) and normalized against the expression of GAPDH or U6. The program was performed at 95°C for 10 min, 95°C for 10 sec plus 60°C for 30 sec for 40 cycles. For the melting curve evaluation, the temperature was slowly increased from 60°C to 97°C, and 5 acquisitions per °C were performed continuously. All samples were analyzed in triplicate. Relative expression was calculated using the comparative threshold cycle (Ct) method and was indicated as n-fold change = −(ΔCt_KO − ΔCt_WT). The primers for Tnfaip3, NF-κB and GAPDH were: for Tnfaip3, 5′-CAGCACCTAAG CCAACGAGT-3′ and 5′-TGGACCTGTCAATGTGTTCG-3′; for NF-κB, 5′-AGCTTATGCCGAACTTCTCG-3′ and 5′-GAC TCCGGGATGGAATGTAA-3′; for GAPDH, 5′-GCAAGG TCATCCCAGAG-3′ and 5′-AAGTCGCAGGAGACAAC-3′; for miR-694 and U6, the miRNA specific primers were respectively: 5′-CTGAAAATGTTGCCTGAAG-3′ and 5′-CAAGG ATGACACGCAAATTCG-3′, whereas the reverse primer was the manufacturer-provided miScript universal primer.

Liver tissues from 2 one-month male Lass2-KO mice and 2 one-month male WT mice were respectively subjected to the experiments of Mouse OneArray (MOA 2.0 ver.) and Mouse and Rat miRNA OneArray (MRmiOA 4.0 ver.) using Phalanx microarray platform by Oebiotech Co. (Shanghai, China). Briefly, RNA was extracted from liver tissues with TRIzol reagent (Invitrogen), whose quantity and purity were assessed using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA). The purity and integrity of each extracted RNA met the requirements: A₂₆₀/A₂₈₀ >1.6, A₂₆₀/A₂₃₀ >1 and RNA integrity number (RIN) value >5. Small RNA fraction indicated the abundance of RNA <200 nt compared with the overall RNA area from Agilent RNA 6000 Nano assay and was acceptable for the miRNA assay. Possibility of genomic DNA contamination was excluded by gel electrophoresis. Two micrograms of RNA from each group was respectively converted into cyanine-5 labeled target cRNA, hybridized to either Mouse OneArray or Mouse and Rat miRNA OneArray by the Affymetrix GeneChip fluidics station 450, and scanned with an Affymetrix GeneChip scanner 3000 7G. After normalization, differentially expressed mRNAs or miRNAs were established at log₂ |Fold change| >1 and P<0.05.

For advanced data analysis, all biological replicates were pooled and calculated to identify differentially expressed genes based on the threshold of fold change and the P-value. The correlation of expression profiles between biological replicates and treatment conditions was demonstrated by unsupervised hierarchical clustering analysis. A subset of genes was selected for clustering analysis. An intensity filter was used to select genes where the difference between the maximum and minimum intensity values exceeded 4,000 among all microarrays. For this microarray project, the number of genes clustered was 272 genes. According to previously selected differentially expressed gene lists, Gene Ontology (GO) analysis was performed by Oebiotech Co. Targetscan 5.1 was utilized to predict miR-targeting mRNA. mRNA-miRNA integration analysis demonstrated potential mRNA targets with inverse expression alterations as their regulatory miRs displayed in the mRNA microarray or miRNA microarray.

Data were analyzed by the Student’s t-test. P<0.05 was considered to indicate a statistically significant difference, and P≤0.01 and P≤0.001 are indicated by relevant symbols in the figures and legends. qPCR and western blot analyses were repeated three times.

The average ratio of liver weight/body weight of the Lass2-KO mice was higher than the ratio in the control WT mice (Fig. 1A). Compared to the liver tissues from the WT mice, the hepatocytes of the Lass2-KO displayed abundant vesicles (Fig. 1B).

The production of ALB in the liver was attenuated in the Lass2-KO mice (Fig. 2A). The levels of ALT, AST and LDH in serum were respectively elevated in the Lass2-KO mice, indicating abnormal liver function in the Lass2-KO mice (Fig. 2B–D), in accordance with the structural injury of the Lass2-KO hepatocytes.

mRNA and miRNA microarray results showed that over 600 mRNA were markedly upregulated and over 700 genes were downregulated; whereas 13 miRNAs were markedly upregulated and 31 miRNAs were downregulated (Table I). According to the GO analysis, ‘response to wounding’ and ‘inflammatory response’ were among the top-10 altered pathways (Table II). miRNA-mRNA integrated analysis identified 4 upregulated and 3 downregulated miRNAs and their respective negatively controlled target genes (Table III).

The level of miR-694 was downregulated in the Lass2-KO liver tissue, as confirmed by qPCR (Fig. 3).

Tnfaip3, one of the markedly upregulated mRNAs in the Lass2-KO liver tissues, which is also a putative target gene of miR-694, was confirmed by qPCR (Fig. 4A). Its negatively controlled NF-κB was found to be downregulated (Fig. 4B).