ABSTRACT
As confusion mounts over RNA isoforms involved in phenotypic plasticity, aberrant CpG methylation-mediated disruption of alternative splicing is increasingly recognized as a driver of intratumor heterogeneity (ITH). Protease serine 3 (PRSS3), possessing four splice variants (PRSS3-SVs; PRSS3-V1-V4), is an indispensable trypsin that shows paradoxical effects on cancer development. Here, we found that PRSS3 transcripts and their isoforms were divergently expressed in lung cancer, exhibiting opposing functions and clinical outcomes, namely, oncogenic PRSS3-V1 and PRSS3-V2 versus tumor-suppressive PRSS3-V3, by targeting different downstream genes. We identified an intragenic CpG island (iCpGI) in PRSS3. Hypermethylation of iCpGI was mediated by UHRF1/DNMT1 complex interference with the binding of myeloid zinc finger 1 (MZF1) to regulate PRSS3 transcription. The garlic-derived compound diallyl trisulfide cooperated with 5-aza-2'-deoxycytidine to exert antitumor effects in lung adenocarcinoma cells through site-specific iCpGI demethylation specifically allowing MZF1 to upregulate PRSS3-V3 expression. Epigenetic silencing of PRSS3-V3 via iCpGI methylation (iCpGIm) in BALF and tumor tissues was associated with early clinical progression in patients with lung cancer but not in those with squamous cell carcinoma or inflammatory disease. Thus, UHRF1/DNMT1-MZF1 axis-modulated site-specific iCpGIm regulates divergent expression of PRSS3-SVs, conferring nongenetic functional ITH, with implications for early detection of lung cancer and targeted therapies.
ABSTRACT
Background: Alternative splicing commonly occurs in cancer cells and many cancer specific splice variants have been reported as potential candidate biomarkers of the disease. We have studied human tissue Kallikrein 7 (KLK7) mRNA expression profile in breast cancer patients of our region. KLK7 is member of a multi-gene family consisting of 15 members (KLK1-KLK15). Methods: We optimized touch down nested PCR method for the amplification of KLK7 isoforms/variants. Various bioinformatics tools were used for sequence analysis, identification of splicing pattern and prediction of encoded proteins. Results: We observed an unusual splicing event consisting of exon 3 (E3) truncation at 3' end (by 124 nucleotides), exon 4 (E4) exclusion and exon 5 (E5) truncation at 5' end (by 33 nucleotide) in 2 normal breast tissues, one obtained from invasive ductal carcinoma grade II patient and other collected from mammary dysplasia patient. Moreover, 3 other KLK7 mRNAs (KF963190, KF963191, and KF963193) expressed in breast cancer were noticed to exhibit single nucleotide polymorphism (SNPs). Bioinformatic analysis revealed that the alternatively spliced mRNA (KF963192) will potentially encode a truncated and non-functional protein. Similarly although encoded proteins have considerable homology with normal hK7 protein, SNPs seem to cause great variations in pIs, structures and molecular weights of encoded proteins. Conclusions: There is need to further explore the impact of the unique splicing event, SNPs and characterize these population specific mutations and their possible role in the pathogenesis of breast cancer (AU)
Subject(s)
Male , Female , Pregnancy , Infant, Newborn , Infant , Child, Preschool , Child , Adolescent , Adult , Middle Aged , Aged , Breast Neoplasms/genetics , Biomarkers, Tumor , Down-Regulation , Protein IsoformsABSTRACT
Objective To study the expression of plasma membrane Ca2 + -ATPase isoforms 1 -4 and the splice variants at sites A and C in the neonatal rat vestibular organ.Methods Ten rats at postnatal 2 days (P2 ) were decapitated and their vestibular organs (macula utriculi and macula sacculi)were isolated.The total proteins of the vestibular organs were extracted.The expression of PMCA1-4 splice variants at sites A and C was detected by RT-PCR.Results The splice variants of PMCA1-4 at sites A and C in macula utriculi and macula sacculi of neo-natal rat vestibular organs were PMCA1x/b,PMCA2w/(a,b),PMCA3z/(a,b,c)and PMCA4 (x,z)/b.Conclusion The splice variants at sites A and C among PMCA1,PMCA2,PMCA3 and PMCA4 were different in the vestibu-lar organs of neonatal rats,which could be explained that macula utriculi and macula sacculi had different require-ments of Ca2 + turning for these PMCA isoforms.
ABSTRACT
The anti-apoptotic effect of (-)-epigallocatechin-3-gallate (EGCG) during unilateral testicular torsion and detorsion (TT/D) was established in our previous study. In mice, the smallest inhibitor of apoptosis, survivin, is alternatively spliced into three variants, each suggested to have a unique function. Here, we assessed how EGCG exerts its protective effect through the expression of the different survivin splice variants and determined its effect on the morphology of the seminiferous tubules during TT/D. Three mouse groups were used: sham, TT/D+vehicle and TT/D treated with EGCG. The expression of the survivin variants (140 and 40) and other apoptosis genes (p53, Bax and Bcl-2) was measured with semi-quantitative RT-PCR. Histological analysis was performed to assess DNA fragmentation, damage to spermatogenesis and morphometric changes in the seminiferous tubules. In the TT/D+vehicle group, survivin 140 expression was markedly decreased, whereas survivin 40 expression was not significantly different. In parallel, there was an increase in the mRNA level of p53 and the Bax to Bcl-2 ratio in support of apoptosis induction. Histological analyses revealed increased DNA fragmentation and increased damage to spermatogenesis associated with decreased seminiferous tubular diameter and decreased germinal epithelial cell thickness in the TT/D+vehicle group. These changes were reversed to almost sham levels upon EGCG treatment. Our data indicate that EGCG protects the testis from TT/D-induced damage by protecting the morphology of the seminiferous tubules and modulating survivin 140 expression.
Subject(s)
Animals , Mice , Apoptosis , DNA Fragmentation , Epithelial Cells , RNA, Messenger , Salicylamides , Seminiferous Tubules , Spermatic Cord Torsion , Spermatogenesis , TestisABSTRACT
Array of new targets for investigation as cancer therapeutics has great potential to grow as new splice-variants are identified and characterized in cancer cell-lines and tumor samples. Tumor-specific splice variants are being discovered at an increasing rate and their functions are also investigated in cancer progression. The tumor-specific splice variants whose expression patterns and activities are successfully characterized may become attractive targets for ablation or splicing modification. The extreme specificity of their expression suggests that a variant-specific treatment may allow for targeting of cancerous cells with minimal impact to healthy tissues. Clinical investigation of applying antisense oligonucleotides to down-regulate mRNAs that contribute to cancer cell survival and to modify splicing patterns in muscular dystrophy has shown promising results. These results show that antisense therapy may be applied effectively and safely in humans. As these treatment strategies continue to improve and novel tumor-specific splice-variants are identified, modification of splicing patterns will become an important field of investigation to develop more effective and safe cancer therapies.
ABSTRACT
Survivin variants specific real time quantitative RT-PCR was developed to analyze their expression in 53 paired cancer and para-cancerous tissues, and the expression of the wild-type survivin protein was detected by immunohistochemistry. The results showed that survivin mRNA and protein were expressed in gastric cancer and para-cancerous tissues. The survivin-2B was dominantly expressed in para-cancerous tissues, whereas the survivin-△Ex3 was more frequently detected in cancer tissues. The positive rate of survivin-2a was 100% in both cancer and para-cancerous tissues,but its relative transcript expression level was not significantly increased in cancer tissues in comparison with para-cancerous tissues. The correlation analysis revealed that the expression of survivin-2a mRNA was significantly associated with that of total survivin (rs=0.4178, P=0.0018),whereas inversely to that of survivin-/EX3 (rs=-0.4506, P=0.0007). It was suggested that survivin-2a may act as an antagonist of survivin-△EX3. The balance between antiapoptotic survivin iso-forms and nonantiapoptotic ones may play an important role in tumorigenesis and tumor progression. Promising value is hinted to analyze survivin and its variants in tumor early diagnosis and distinguishing malignant tumors from benign ones.