The Research On The Expression Of A Bpes Rela Ted Gene SOX14 In Cervical Cancer Cell Lines

Background:The Blepharophimosis Ptosis Epicanthus Inversus Syndrome(BPES), characterized by narrowed horizontal palpebral aperture, ptosis and a skinfold extending from the lower eyelid, is a disease of autosomal dominant inheritance, in which the disease locus had been located on 3q23. Horizontal palpebral fissure measures 25 to 30 mm in normal adult. But as to BPES patients, it is usually 20 to 22 mm in length. Ptosis in BPES, which is bilateral and symmetric, is due to poor levator function from dysplasia of the levator aponeurosis. In addition to infertility found in affected females of type I, BPES patients also have several other extraocular findings, including microcephaly and limb deformity. Almost all BPES cases with microcephaly and limb deformity have interstitial deletions, which include 3q23. SOX14 was located at human chromosome 3q23, expressing a transcriptional repressor which has shown a high level of conservation among vertebrates. In recent years many foreign scientists have found mutations in SOX14, regarding it as a putative gene associated with microcephaly and limb deformity in BPES patients. In this study, we intended to research the expression trait of SOX14 in several cervical cancer cell lines in order to see whether SOX14 may have the potential to serve as a marker of cervical cancer.Objective: In this study, we intended to research the expression trait of SOX14 in several cervical cancer cell lines by RT-PCR, Real-Time PCR and western blotting, in order to see whether the SOX14,a BPES related gene, may have the potential to serve as a marker of cervical cancer.Methods:The total RNA was extracted by Trizol from 293 T, Hela, HT-3, Caski and SiHa cells, and the extracted RNA was qualified by formaldehyde agarose denaturing gel electrophoresis. Then RT-PCR was performed with the oligo(dT)18 primers from the Fermentas RT-PCR kit, which produced cDNA of the total mRNA. After that, PCR was run with the designed SOX14-specific primers and the new reverse transcripted cDNA, so as to test whether SOX14 was expressed in these cervical cancer cell lines. The results were identified through DNA gel electrophoresis. Real-time PCR techniques were performed to test the difference of SOX14 expression level in different cervical cancer cells. Amplification quality was demonstrated by amplification curve and solubility curve. Non-malignant cell line 293 T was used as a control to compare the mRNA expression level of SOX14 in the other 4 cervical cancer cell lines. Finally, we detected whether the SOX14 protein has been expressed and compared the relative expression level of it by western blot.Results:The result of formaldehyde agarose denaturing gel electrophoresis showed that the extracted total RNA of high-quality was suitable for RT-PCR. Agarose gel electrophoresis visualized that mRNA of SOX14 were expressed in all cervical cancer cell lines, indicating that the SOX14 gene of these cell lines are expressing at mRNA level. Real-time PCR showed that SOX14 mRNA in cervical cancer cell lines was far less than beta-actin the internal reference, compared with in 293 T. SiHa cells have the highest expression of SOX14 mRNA among these four cell lines, while Caski cells have the fewest one. The results of western bloting are substantialy in agreement with the results of real time PCR. Among these cell lines, Caski cells expressed SOX14 lowest in protein level and the corresponding band of which was unsharp in westernbloting figure.Conclusions:In this study, we confirmed that SOX14 was expressed in all selected cervical cancer cells. Surprisingly, we found the expression of SOX14 in 293 T at the same time. Therefore, we planed to take 293 T as a control cell lines so as to compare the relative expression level of SOX14 in several cervical cancer cell lines.Real-time PCR showed that SiHa cells had the highest expression of SOX14 mRNA, while Caski cells had the fewest one. However, westernblot revealed that Hela, SiHa and HT-3 had no difference in SOX14 expression at protein level, despite Caski had the fewest expression. Because SOX14 is only expressed in certain spots of certain stages on the embryonic development, the results of our study primarily indicated that SOX14, as an BPES associated gene, may become a potential marker in the process of cervical cancer oncogenesis.