| Malignancy always appears to be a common and frequently occurring disease severely harmful for people’s health. Gastric carcinoma is one of malignancies which have the secondary mortality rate in cancer. Surgery combined with radiation and chemotherapy is a major clinical treatment to gastric tumor. However, primary or secondary drug resistance is often produced in patients with gastric cancer in the setting of chemotherapy, and even progresses into multidrug resistance with cross tolerance to diverse agents, thus finally leading to chemotherapy failure.The complicated mechanism of drug resistance in cancer ranging from overexpression of drug efflux pump protein family and multidrug resistance protein family (MDR), mutation or alteration of drug targets to enhancement of the cellular repair system and attenuation of apoptosis,etc, which are,as yet,unclearly demonstrated.It is well documented that drug resistance in malignancy including gastric carcinoma could be attributed to combinational or synergetic effect.MicroRNAs (miRNAs) are single-stranded RNAs (ssRNAs) of19-25nucleotides in length that are generated from endogenous hairpin-shaped transcripts. MiRNAs widely exist in animals and plants which function by base pairing with target mRNAs’3’-UTR, which mediates mRNA cleavage or translational repression. MiRNAs are closely associated with drug resistance,which are supported by the markedly differential expression of miRNAs between sensitive and resistance cells, and the regulation of target genes by miRNA contributing to resistance in cancer cells. In mammals, there are16members,14large catalysis subunit members,1small regulatory subunit member, and1endogenous inhibitor in the calpain family. Calpain small submit1(CAPNS1) is the regulatory subunit targeting calpainl and calpain2, the members of calpain family. The calpain family affects proliferation,apoptosis, migration, differentiation, autophagy in cancer cells and can directly cleave caspase family members then induce apoptosis. As one of the first line chemotherapy regimens, cisplatin (DDP) is widely applied in postoperative chemotherapy. Two cell lines resistant to cisplatin, SGC-7901/DDP and BGC-823/DDP, were induced in our study. Cisplatin resistance was developed by exposure of parental SGC-7901and BGC-823cells to DDP. On the basis of identification of drug resistance phenotype and cell drug resistance index, micro RNA microassays were performed to analyze the different expression of miRNA between sensitive and resistance cells. Two-dimensional gel electrophoresis in combined with mass spectrometric analyses screened the differentially expressed miRNA and proteins between them.Pooled analyses of the data from two microassays were conducted incorporated with comparative analysis of miRNAomes and proteomes.All the results have been confirmed by RT-PCR Western Blot, Dual Luciferase Reporter assay,which revealed the upstream regulation role of miR-99a and miR-491in CAPNS1expression and their impact on cisplatin resistance in4gastric cancer cell lines.The mechanism of the indicated impact was preliminarily explored, which showed calpainl and calpain2,regulated by CAPNS1, were implicated in cisplatin resistance in gastric cancer by cleavage of the downstream proteins caspase3and PARP1.Objective:Resistance-related miRNAs and their target genes were screened by high throughput chips on miRNAomes and proteomes level and the mechanism of miRNAs modulation of cisplatin resistance in gastric cells was explored to provide novel ideas for rescuing resistance to chemotherapy on individualizedbasis.Methods:The cisplatin resistant sublines SGC-7901/DDP and BGC-823/DDP were induced previously.The Affymetrix miRNA2.0microarrays were performed on the sensitive and resistant cells respectively and clustering analysis was carried out with cluster3.0to detect the miRNA expression profile. With two-dimensional gel electrophoresis (2-D GE) and mass spectrometry (MS), the differentially expressed proteins from2pairs of cell lines were identified and significant differences in80protein spots were found. The combination of bioinformatics website MicroCosm Targets Version5and miRNA expression microarrays were conducted to screen and predict miRNA affecting the80proteins differentially expressed. The differential expression of miRNA were verified with application of RT-PCR and dual luciferase reporter gene assay was used for validation of the direct binding of miRNA and target gene3’-UTR. The clone formation assay following respective transfection of the two groups of cells by miRNA mimics and inhibitor upon Cisplatin was done while the colonies were counted under a microscope.Immunofluorescence was employed as a tool for detection of TUNEL-positive cells exposed to diverse doses of cisplatin after transfection. To identified the altered expressions of CAPNS1and calpainl/2,members of calpain family coupled with caspase3and PARP1in the2groups of cells, Western blot was performed,24h prior to addition of gradient dose of cisplatin. HA-CAPNS1and si-CAPNS1, the CAPNS1up-regulated and knockdown plasmids, were transfected into resistant and sensitive cells respectively before cisplatin treatment. The Expressions of downstream caspase3and PARP1were assayed by Western blot. Finally, the phenotypes of the CAPNS1over-expressing and silencing cells transfected with miRNA mimics and inhibitor were verified adopting clone formation assay and Immunofluorescence.Results1. A total of68miRNAs exhibiting more than2-fold discrepancy were found in miRNA expression profiling analysis of SGC-7901and SGC-7901/DDP, including41upregulated and27downregulated in resistant cells. Comparison of BGC-823and BGC-823/DDP showed94miRNAs with more than2-fold expression difference, among which41were upregulated and27downregulated in resistant cells.7miRNAs were simultaneously upregulated and6downregulated in the two resistant cells lines.2. Screening of two groups by two-dimensional gel electrophoresis revealed2547differential protein spots, including1236in SGC-7901and SGC-7901/DDP and1311in BGC-823and BGC-823/DDP. MALDI-TOF-MS analysis of80screened differential expression protein spots was conducted and77GI from NCBI protein database. Compared with SGC-7901,16of them in SGC-7901/DDP were detected as more than2-fold up-regulated expression, while20down-regulated. In contrast,18in BGC-823/DDP were markedly (more than2-fold) upregulated and23downregulated relative to BGC-823.3. miR-99a and miRNA-491up-regulated concurrently in two resistant cell lines were confirmed by Q-PCR compared with sensitive cells. The levels of miR-99a and miRNA-491expression were increased by4.98±0.51times (n=3, P<0.01) and2.60±0.54folds(n=3, P=0.07), respectively in SGC-7901/DDP,while3.83±0.18times (n=3, P<0.01)å'Œ2.16±0.56times (n=3, P=0.18) in BGC-823/DDP. On the contrary, miR-27a and miR-424exhibited low expressions synchronously in resistant cells, of which were reduced by0.52± 0.01times(n=3,P=0.11) and0.61±0.07times (n=3,P=0.13) in SGC-7901/DDP while0.58±0.10times(n=3,P=0.28) and0.25±0.002times(n=3, P<0.01) in BGC-823/DDP.4. The data from two-dimensional gel electrophoresis (2-D GE) and mass spectrometry (MS), which was verified by Western blot, showed remarked low expression of CAPNS1accompanied by calpainl and calpain2, the catalytic subunit of calpain, in both resistant cells. Reversely, highly expressed CAPNS1was observed in sensitive cells whereas calpainl and calpain2displayed lower levels of expression than resistant cells.5. Bioinformatics website MicroCosm Targets Version5and miRanda was utilized for miR-99a and miR-491potential target prediction by searching for conserved sites that match the seed region of each miRNA, which revealed a shared target gene CAPNS1.Moreover,The possible target mRNAs3’-UTR have a overlapped sequence with the potential binding sites of miR-99a and miR-491. Intriguingly, CAPNS1was one of the previously differentially-expressed proteins screened by two-dimensional gel electrophoresis (2-D GE) and mass spectrometry (MS) and confirmed by Western blot.6. The findings in Luciferase reporter assays indicated that CAPNS1is a direct downstream target for miR-99a and miR-491by its3’-UTR(3’-Untranslated Regions). In addition, the mutant of binding site led to failure of binding of miRNA to CAPNS1mRNA.7. The elevated levels of miR-99a and miR-491by transfection respectively contributed to increasing level of CAPNS1protein in sensitive cells, but inhibition of miR-99a and miR-491expressions was implicated in reduced expression of CAPNS1. Silencing and overexpressing the indicated miRNAs simultaneously failed to neither attenuate competitively nor potentiate the regulation of CAPNS1in two groups of paired cells.8. Colony formation assay demonstrated that colony formation efficiency were enhanced in sensitive cells that overexpressed miR-99a or miR-491followed by treatment with cisplatin while reduced in resistant counterparts that silencing miR-99a or miR-491before cisplatin exposure. Likewise, after subsequent cisplatin addition, elevated colony formation efficiency was found in sensitive cells with knockdown of CAPNS1and decreased in CAPNS1-upregulated sensitive cells.9. Immunofluorescence microscopy exhibited that number of TUNEL-positive cells,after exposing to cisplatin, was decreased in sensitive cells with depletion of miR-99a or miR-491and increased in resistant cells with overexpression of miR-99a or miR-491. As regards to target gene CAPNS1, CAPNS1-knockdown sensitive cells by SiRNA showed reduced TUNEL-positive cells while the number of CAPNSl-upregulated resistant cells were spot elevated in the setting of cisplatin.10. CAPNS1expression increased with cisplatin dose growing in4cell lines sequentially exposed to increasing concentrations of cisplatin for24h.Besides, calpainl/2, the large catalytic subunit of calpain, appeared increased thus undergoing cleavage of its target caspase3responsible for successive cleavage of PARPl,the effector during apoptosis, which is characterized by elevated level of cleaved PARP1. Reduced level of calpainl/2suppressed the cleavage of caspase3and PARP1in sensitive cells devoid of CAPNS1treated with cisplatin. Furthermore, the rising level of CAPNS1by transfection elicited increasing expression of calpainl/2partnered with the formation of cleaved caspase3and PARP1upon cisplatin stimulation in resistant counterparts.Conclusion: Discrepant expressions of miR-99a,miR-491andCAPNS1were observed between sensitive and resistant cells and the miRNAs appeared direct regulator of CAPNS1,however,in an uncompetitive or unsynergetic manner.The precise regulation of miRNA-CAPNS1-calpainl/2-caspase3-PARP1were related to cisplatin resistance in gastric cancer. It is first documented that the CAPNS1-regulatory role of miR-99a and miR-491as well as the function of calpain family members in cisplatin resistance of gastric cancers.The study expands the understanding of mechanism of drug resistance in gastric neoplasias and deepens our insight into the association of miRNA with chemotherapy resistance.Furthermore,it provides novel potential targets for clinical screening and resistance intervention. |
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