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Expression Patterns And Functional Studies Of Kiaa1217 Gene Selective Splice Variants In Tumor Cells

Posted on:2018-09-13Degree:MasterType:Thesis
Country:ChinaCandidate:L ChuFull Text:PDF
GTID:2404330515969431Subject:Cell biology
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Gene alternative splicing is an important mechanism for controlling gene expression and producing protein functional diversity and plays a very important role in development,differentiation and cancer.Abnormal gene splicing can lead to genetic diseases and even cancer.A large portion of human cancer relative genes is associated with selective splicing of genes,and cancer-specific splice variants have important application value in cancer diagnosis and as specific therapeutic targets,which makes study of gene selective splicing in cancer a hot spot today.Studies have shown that gene-selective splicing can lead to cancer in a variety of ways,so in-depth study of splice variants in the occurrence and development of cancer may lay a theoretical basis for early diagnosis and treatment of cancer and provide new ideas for seeking new therapeutic methods.Kiaa1217 is a relatively new gene and only a few reports showed its functional role mainly involve the disc herniation and that it could exist as a fusion gene.In the previous study,we compared the expression of the gene splice variants between 9 kinds of cancer tissues and corresponding normal tissues using the second generation sequencing analysis technique.We found that Kiaa1217 gene splicing variant differentially expressed in liver cancer,lung cancer and breast cancer.However,the expression pattern of the Kiaa1217 gene variants in tumor cells and their roles in the development and progression of tumors remains unclear.In this study,we identified seven variants of the Kiaa1217 gene according to the NCBI database,and used RT-PCR to distinguish the different variants of the Kiaa1217 gene by designing different primers.Then we examined the expression of variants in different cell lines and carried out related functional studies.The main results are as follows:1.The expression of Kiaa1217 gene splice variants was analyzed by RT-PCR in a variety of cell lines.It was found that the expression pattern of Kiaa1217 splice variants was different in different types of tumor cells or in different tumor cell lines from the same type;2.By using RT-PCR combined with DNA sequencing,we first discovered a novel Kiaa1217 gene variant named V9 which is very similar to the variant V6,but lack of 78 bp long exon 18;3.The Kiaa1217 gene was identified to be probably present as a fusion gene in human ovarian cancer cell SKVO3 by RT-PCR analysis and PCR amplification of the Kiaa1217 genomic fragment using different primers4.Compared with normal human hepatocytes L02,the Kiaa1217 V4/5 variants in human Bel-7402 liver cancer cells was highly expressed in Bel-7402.Therefore,by using siRNA technology to interfer with the expression of Kiaa1217 V4/5 and total Kiaa1217 in Bel-7402 cells,we explored the biological function of Kiaa1217 and its V4/5 variants in hepatocellular carcinoma.(1)Taking MTT method,we found the viability of Bel-7402 cells was significantly inhibited when expression of Kiaa1217 V4/5 and total Kiaa1217 was down-regulated by siRNAs.(2)Combined use of DAPI staining to observe the apoptotic bodies and Annexin V-FITC / PI double staining for FACS analysis of apoptotic cells proved that the decreased expression of total Kiaa1217 and its V4/5 variant promoted the apoptosis of Bel-7402 cells.Further Western Blot analysis showed that downregulation of total Kiaa1217 and its V4/5 variants increased the levels of activated cleaved-caspase 9 and caspase-3 while decreased the expression ratio of Bcl-2/Bax,suggesting that decreased expression of Kiaa1217 V4/5 or total Kiaa1217 could induce the apoptosis of human hepatocellular carcinoma Bel-7402 cells by activating the mitochondrial apoptosis pathway.(3)The combination of Brdu incorporation,soft agar clone formation assay and plate cloning assay showed that the decrease in the expression of total Kiaa1217 and its V4/5 variants had no effect on the proliferation of Bel-7402 cells,and further flow cytometry analysis of cell cycle and Western Blot analysis of cycle-related proteins also demonstrated the above conclusion;(4)Using the scratch test and transwell chamber,we demonstrated that the migration ability of Bel-7402 was significantly detroyed after decreasing expression of total Kiaa1217 and its V4/5 variants.When the expression of EMT-related marker proteins were further analyzed by Western Blot,we found the down-regulation of Kiaa1217 and its V4/5 variants promoted E-cadherin expression but inhibited the expression of N-cadherin,vimentin and MMP9.This suggests that the EMT process is inhibited,which may be one of the mechanisms by which down-regulated Kiaa1217 and its V4/5 variants declined the migration ability of Bel-7402 cells.In conclusion,this study first analyzed the expression pattern of Kiaa1217 gene alternative splicing variants in tumor cells and for the first time,revealed the biological effects of Kiaa1217 and its variants in tumor cells,which may provide new ideas for early diagnosis and treatment of tumors.
Keywords/Search Tags:Kiaa1217, gene alternative splicing, hepatocarcinoma, apoptosis, cell migration
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