Reagents
Dex was dissolved in dimethyl sulfoxide (DMSO) as a stock solution (200 mM). All reagents were from Sigma-Aldrich, St. Louis, MO, USA.
HLEC culture
The HLEC line B-3 was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The product number was ATCC CRL-11421. The cells were maintained in culture with Dulbecco’s modified Eagle’s medium (DMEM; Gibco Co., Gaithersburg, MD, USA) with 20% fetal calf serum, 100 U/mL penicillin, 0.1 mg/mL streptomycin and 2 mM L-glutamine in white six well plates with a transparent bottom (Corning, Sunnyvale, CA, USA) with approximately 105 cells per well to yield a confluent monolayer. The treatments were the same for all assays.
Establishment of alphaA- and alphaB-crystallin overexpression in cells and detection
The establishment of stable overexpression of αA- and αB-crystallin in HLECs was as follows: The cDNA of αA- and αB-crystallin in the cell was used as a template, and was amplified by primer PCR and cloned to the screen Pcdna3.1-myc-his (abbreviated to pcDNA3.1) blank carrier selected. Digestion and sequencing confirmed whether the construction of αA- and αB-crystallin expression plasmids was successful.
We added 2 µ l plasmid into competent cells of E. coli. After successful transformation, observe the growth of bacteria and select monoclonal colonies. A large number of plasmids were successfully extracted from the colonies and transfected into hleb3 cells. After 4–6 weeks, high concentration (1000 μg/μL) G418 was used to screen the monoclonal cell lines expressing foreign genes. Half of the medium was renewed every 24 hours until the cells grew stably. Western blot was used to detect the expression of αA- and αB-crystallins. Purified α A- and αB-crystallin were stored at -80°C. After that, the following experiments were carried out on the obtained stable cell lines: The effect of αA- and αB-crystallin expression on phenotype, and stable transfection of α A- and αB-crystallin and their different domains using EGFP. The expression of EGFP was observed under a fluorescence microscope. Images were obtained by fluorescence microscopy and stored in the computer. 1 × 106 cells were subcultured in a 100 mL culture flask. After culture at four concentrations for four corresponding time periods, the cells were rinsed three times with D-Hank's solution. The cells in the control group were cultured with DMEM without Dex. Other methods were the same as those in the experimental group.
Cell treatment
The HLEC line B-3 obtained from ATCC was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco) with 20% fetal calf serum, 100 U/mL penicillin, 0.1 mg/mL streptomycin and 2 mM L-glutamine in white six well plates with a transparent bottom (Corning) with approximately 105 cells per well to yield a confluent monolayer. The treatments were the same for all assays. Three groups were used for each assay: the pc DNA3.1- HLECs group, the pc DNA3.1-αAC- HLECs group and the pc DNA3.1-αBC- HLECs group. In the Dex +pc DNA3.1- HLECs group, cells were treated with 0.1 μmol, 1 μmol, 10 μmol and 100 μmol Dex for 12 h, 24 h, 36 h, and 48 h. In the Dex + pc DNA3.1-αAC and αBC-HLECs groups, cells were treated with 0.1 μmol, 1 μmol, 10 μmol, and 100 μmol Dex for 12 h, 24 h, 36 h, and 48 h.
The expression of nine proteins detected by western blot
Protein samples were collected from cultured cells in the three groups. After treatment with drugs, the cells were treated with 5 SDS PAGE buffer. Protein concentrations were determined using the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL, USA). Samples were electrophoresed on 12% Tris-glycine polyacrylamide gels using commercial apparatus (Bio-Rad Mini-PROTEAN Tetra cell) and then blotted onto a nitrocellulose membrane (Pall-Gelman) using a Transblot cell (Bio-Rad, Hercules, CA, USA). Membranes were blocked with 3% BSA and probed with nine primary antibodies and corresponding secondary antibodies using standard protocols. For HSP60 protein ,membranes were blocked in 3% BSA and probed with antibody (MA3-013, 1:1000, Invitrogen Antibodies) and secondary antibodies (Goat anti-mouse Ig-G, Beyotime Institute of Biotechnology) ;For Bid protein ,membranes were blocked in 3% BSA and probed with antibody (MA1-13043, 1:1000, Invitrogen Antibodies) and secondary antibodies (Goat anti-mouse Ig-G, Beyotime Institute of Biotechnology); For Survivin protein, Membranes were blocked in 3% BSA and probed with antibody (MA5-15077, 1:1000, Invitrogen Antibodies) and secondary antibodies (goat anti-rabbbit Ig-G, Beyotime Institute of Biotechnology) ;For p53 protein, Membranes were blocked in 3% BSA and probed with antibody (7157-MSM3-P1, 1:1000, enQuire BioReagents) and secondary antibodies (Goat anti-mouse Ig-G, Beyotime Institute of Biotechnology).For Smac/DIABLO protein, Membranes were blocked in 3% BSA and probed with antibody (PA1-41007, 1:1000, Invitrogen Antibodies) and secondary antibodies (Goat anti-mouse Ig-G, Beyotime Institute of Biotechnology);For cytochrome C protein, Membranes were blocked in 3% BSA and probed with antibody (710627, 1:1000, Invitrogen Antibodies) and secondary antibodies (Goat anti-mouse Ig-G, Beyotime Institute of Biotechnology);For NF-К B protein, Membranes were blocked in 3% BSA and probed with antibody (PA5-27617, 1:1000, Invitrogen Antibodies) and secondary antibodies (Goat anti-rabbit Ig-G, Beyotime Institute of Biotechnology);For Fas protein, Membranes were blocked in 3% BSA and probed with antibody (MA5-14882, 1:1000, Invitrogen Antibodies) and secondary antibodies (Goat anti- rabbit Ig-G, Beyotime Institute of Biotechnology).All protein bands were visualized by diaminobenzidene (DAB, Tiangen Biotech Co. LTD) stain using a gel documentation apparatus (FUJIFILM LAS-3000).The membranes visualized by diaminobenzidene (DAB, Tiangen Biotech Co., Ltd) stain using gel documentation apparatus (FUJIFILM LAS-3000).
Blots were reprobed with anti-actin monoclonal antibody(MA5-11869, 1:1000, Invitrogen Antibodies) with Goat anti-mouse IgM secondary antibody (Beyotime Institute of Biotechnology, China), imaged as described above, and densitometric analyses were performed using NIH Image (version 163) with values normalized to the actin signal for each sample and expressed as multiples of increases (or decreases) relative to control samples (available at http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD, USA). Following analysis, we observed the most significantly expressed protein, the exposure factors and cell types.
LncRNA and mRNA chip scanning
Based on the results of western blotting, the RNA samples of HLECs (α A-crystallin overexpressed) were exposed to 0.1, 1, 10, and 100 μmol Dex for 36 h and 48 h; HLECs (αB-crystallin overexpressed) were exposed to 10 and 100 μmol Dex for 36 h and 48 h; HLECs were exposed to 0.1, 1, 10, and 100 μmol Dex for 36 h and 48 h and lncRNA and mRNA chip analysis was performed. After identifying the most significant exposure factors and cell types, total RNA was extracted from the cells and scanned by lncRNA and mRNA chips. RNA was extracted using an RNA extraction kit. The concentration of RNA was detected at 260 nm and 280 nm. The RNA in the experimental sample was matched with an Agilent expression microarray (Agilent, Santa Clara, CA, USA). RNA amplification and a single standard kit were used to amplify and label cRNA, which was purified by the RNeasy Mini Kit. Chip hybridization was performed according to the operating instructions of the Agilent gene chip kit in a rolling hybridization furnace at 65°C. At 10R/min and 17 h after rolling hybridization, the sample size of the hybrid cRNA was 1.65 μg, and the film was washed in the washing tank. The chips that completed the hybridization were scanned by the Agilent chip scanner, with feature extraction software 10.7 reads data and gene spring software 11.0. The algorithm was used for normalization. Scanning in the Agilent scanner involved resolution of 3 μm/5 μm, and the scanner automatically scanned once at 100%.
The Agilent SurePrint G3 Human Gene Expression v3 8x60K Microarray (Design ID: 072363) Chip experiment was used to analyze 20 samples. The total RNA of samples was quantified by Nanodrop nd-2000 (Thermo Fisher Scientific, Scotts Valley, CA, USA) and detected using an Agilent Bioanalyzer 2100 (Agilent). After RNA quality was determined, sample labeling, chip hybridization and elution referring to the chip standard were carried out.
Depth analysis showed that lncRNA and mRNA were highly specific compared with the negative control.
The Pearson’s correlation coefficient was obtained by calculating the gene expression correlation analysis, and then the value of expression correlation was determined if it was greater than the pre-set threshold value. The expression correlation was calculated by a statistical method to determine whether it was significantly higher. Pearson’s correlation coefficient, with an absolute value of 0.7, was considered relevant. Less than 0.7 represented a negative correlation, and 0.7 or greater represented a positive correlation. Pearson’s correlation p-value < 0.05 was considered to be significantly related to expression.
For lncRNA (differential expression), all coding bases in the range of 100 K upstream and downstream were searched. Therefore, there was significant co-expression (Pearson correlation calculation) with lncRNA. The lncRNA is likely to regulate the genes that are close to the genome and co-expressed in the expression mode. The “Encyclopedia of DNA elements”, the largest international cooperation project after the “human genome project” Encyclopedia of DNA elements (ENCODE for short) was used in this analysis. We plan to release the transcription factors and their target gene datasets in the latest release results. We calculated the intersection of the coding gene set co-expressed by lncRNAs and the target gene set of the transcription factor/chromatin regulatory complex, calculated the enrichment degree of the intersection using hypergeometric distribution, and obtained the transcription factors significantly related to lncRNAs, in order to identify the transcription factor / chromatin regulatory factors that may play a regulatory role jointly with lncRNAs. Thus, we calculated the coding genes co-expressed by lncRNAs and identified the transcription factors of these genes to identify the transcription factors that may play a regulatory role jointly with lncRNAs.
Screening methods and co-expression patterns of differential gene co-expression were obtained by correlation analysis of computational gene expression. The correlation coefficient was obtained, and it was determined whether the value of the expression correlation was greater than the preset threshold. Statistical methods were used to calculate whether the expression was highly correlated.
To determine whether the expression patterns of lncRNAs were significantly different, the expression patterns of co-expressed coding genes was assessed using a Heat map. The differential integration analysis (Venn analysis) was performed to carry out cross and union analysis of different comparisons of the three groups of cells treated with four concentrations for four time periods to evaluate common or unique differentially expressed genes. Two-way cluster analysis was used to investigate the expression patterns of differential gene expression, and the expression patterns of coding genes that were significantly co-expressed. The distance between two pairs of multiple samples was calculated to form a distance matrix. The two categories with the closest distance were a new category. The distance between the new category and the current category was calculated, and then merged until there was only one category. The direct correlation of samples with the selected differential genes or lncRNA expression was calculated. Generally speaking, the same category of samples can appear in the same cluster through clustering, and the genes or lncRNAs in the same cluster may have similar biological functions. Preconditions for the analysis were the samples and genes or lncRNAs for cluster analysis needed to be specified. Each cluster contained at least three samples, and the number of genes should not exceed 1,000 as much as possible.
Comprehensive analysis of specific lncRNAs and mRNAs
For each differentially expressed lncRNA, the co-expressed coding genes were calculated, and the lncRNA-mRNA relationship pairs with significant correlation were screened out. The function of mature mRNA was used to deduce the function of lncRNA, and the mRNA with a significant correlation with abnormal expression of lncRNA was enriched and analyzed. The principle of functional enrichment analysis is to test the significance of a functional class in a group of interested genes with a hypergeometric distribution pattern. The smaller the p value, the more likely the functional module is related to lncRNA, and the biological process of abnormal expression of lncRNA can be obtained. At the same time, in order to control the situation of significant misjudgment, we introduced the calculation of the false-positive rate (FDR). When FDR < 0.05 it is considered that the functional module may be related to lncRNA. We analyzed the different mRNA molecules after comparing the patients with cataracts and normal controls to identify the upregulated and downregulated lncRNA or mRNA.
Then, GO and KEGG enrichment analyses were carried out to determine the biological functions or pathways that were mainly affected by different genes. The GO database provides professional terms to define the properties of gene products. It includes three categories: biological process (BP) which represents the process of a molecular activity event, including the function of cells, tissues, organs and species, it is often the category with the highest degree of relevance to experimental research problems; cell component (CC) represents the cell or its external environment; molecular function (MF) is the active element describing gene products at the molecular level. GO analysis of the differential genes is carried out to describe the function of the genes. The biological processes, molecular functions and cell components of differential genes, up-regulated differential genes and downregulated differential genes were analyzed. A bar chart was drawn of the items in biological process, cell component and molecular function using the results of GO enrichment analysis, which showed the most significant top 10 GO items in the three categories of GO results in the same chart. A bubble chart was drawn for the top 30 items with the lowest p value in the results of the GO enrichment analysis to show the most significant items in this analysis and their corresponding classification (BP, CC, MF). KEGG is a database for systematic analysis of gene functions, the connection of genomic information and functional information. The number of differential genes included in each GO entry was counted, and the significance of differential gene enrichment in each GO entry was calculated by a hypergeometric distribution algorithm. The result of this calculation will provide a p value of enrichment significance, the lower the value, the more statistically significant it is. According to the results of GO analysis and biological significance, we selected the genes for follow-up study. The KEGG data were used to carry out pathway analysis of differential genes, and the hypergeometric distribution algorithm was used to calculate the significance of the enrichment of differential genes in each pathway. The smaller the p value, the higher the correlation between the corresponding pathway and the differential gene. Using pathway analysis of the differential gene, the pathway significantly related to the differential gene was determined, and indicated which pathway changes may be related to the experimental design conditions.
Real-time quantitative PCR to verify the results of lncRNA chip
The qRT-PCR was used to validate some of the differentially expressed lncRNAs and mRNAs. The primer and probe sequences are listed in Table 1. The qPCR was performed using the SYBR Green Premix DimerEraser kit (TaKaRa, Dalian, China) on the Roche LightCycler 480 Instrument II. The relative gene expression was analyzed using the 2(−ΔΔCt) method as previously described [40]. The significance of differences was analyzed using one-way analysis of variance or the Student’s t-test (unpaired). Multiple comparisons between groups were performed using Tukey’s method. SPSS software, version 19.0 (SPSS, Inc., Chicago, IL, USA) was used to perform the statistical analysis. A p<0.05 was considered to indicate a statistically significant difference.
Establishment of alphaA--crystallin overexpression Primary cell and perform Western blot and Real-time quantitative PCR to verify specific anti apoptotic proteins and related lncRNAs
In order to verify that overexpression of alphaA--crystallin will lead to the increase of the expression of related anti apoptotic proteins and further verify the expression changes of related lncRNAs, we carried out the primary culture of lens epithelial cells and established the primary culture cell line of lens epithelial cells with overexpression of alphaA--crystallin. After different concentrations of dexamethasone for different times, Western blot further verified the expression changes of HSP60, bid, survivin and p53 protein in normal primary cultured lens epithelial cells compared with exposure factors, and the expression of lnc-tmprss15 was detected by real-time quantitative PCR.
The donated eyeballs were removed from the eye bank of the eye hospital of the First Affiliated Hospital of Harbin Medical University, and the lens was separated. The anterior capsular membrane of the lens was removed with capsulorhexis forceps and immediately placed in RPMI-1640 medium. The primary cultured cells can be subcultured when they are 80% - 90% full of the bottom of the bottle. The cells with good growth in the second to fourth generations are used in the experiment. The transformation and cell treatment methods, Western blot and Real-time quantitative PCR methods are identified as described above.