Beyond Death: Unmasking the Intricacies of Apoptosis Escape.

Apoptosis, or programmed cell death, maintains tissue homeostasis by eliminating damaged or unnecessary cells. However, cells can evade this process, contributing to conditions such as cancer. Escape mechanisms include anoikis, mitochondrial DNA depletion, cellular FLICE inhibitory protein (c-FLIP), endosomal sorting complexes required for transport (ESCRT), mitotic slippage, anastasis, and blebbishield formation. Anoikis, triggered by cell detachment from the extracellular matrix, is pivotal in cancer research due to its role in cellular survival and metastasis. Mitochondrial DNA depletion, associated with cellular dysfunction and diseases such as breast and prostate cancer, links to apoptosis resistance. The c-FLIP protein family, notably CFLAR, regulates cell death processes as a truncated caspase-8 form. The ESCRT complex aids apoptosis evasion by repairing intracellular damage through increased Ca2+ levels. Antimitotic agents induce mitotic arrest in cancer treatment but can lead to mitotic slippage and tetraploid cell formation. Anastasis allows cells to resist apoptosis induced by various triggers. Blebbishield formation suppresses apoptosis indirectly in cancer stem cells by transforming apoptotic cells into blebbishields. In conclusion, the future of apoptosis research offers exciting possibilities for innovative therapeutic approaches, enhanced diagnostic tools, and a deeper understanding of the complex biological processes that govern cell fate. Collaborative efforts across disciplines, including molecular biology, genetics, immunology, and bioinformatics, will be essential to realize these prospects and improve patient outcomes in diverse disease contexts.

Investigation of sperm hsa-mir-145-5p and MLH1 expressions, seminal oxidative stress and sperm DNA fragmentation in varicocele.

BACKGROUND: Mechanisms by which varicocele causes infertility are not clear and few studies have reported that some miRNAs show expression alterations in men with varicocele. Recently, sperm promoter methylation of MLH1 has been shown to be higher in men diagnosed with varicocele. This study aimed to assess the potential effects of miR-145, which was determined to target MLH1 mRNA in silico on sperm quality and function in varicocele. METHODS: Sperm miR-145 and MLH1 expressions of six infertile men with varicocele (Group 1), nine idiopathic infertile men (Group 2), and nine fertile men (control group) were analyzed by quantitative PCR. Sperm DNA fragmentation was evaluated by TUNEL and the levels of seminal oxidative damage and total antioxidant capacity were analyzed by ELISA. RESULTS: Our results have shown that sperm expression of miR-145 was decreased in Group 1 compared to Group 2 (P = 0.029). MLH1 expression was significantly higher in Group 2 than the controls (P = 0.048). Total antioxidant level and sperm DNA fragmentations of Group 1 and Group 2 were decreased (P = 0.001 and P = 0.011, respectively). Total antioxidant capacity was positively correlated with sperm concentration (ρ = 0.475, P = 0.019), total sperm count (ρ = 0.427, P = 0.037), motility (ρ = 0.716, P < 0.0001) and normal morphological forms (ρ = 0.613, P = 0.001) and negatively correlated with the seminal oxidative damage (ρ=-0.829, P = 0.042) in varicocele patients. CONCLUSION: This is the first study investigating the expressions of sperm miR-145 and MLH1 in varicocele patients. Further studies are needed to clarify the potential effect of miR-145 on male fertility.

The Interrelationship Between FYN and miR-128/193a-5p/494 in Imatinib Resistance in Prostate Cancer.

BACKGROUND: C-KIT is a receptor tyrosine kinase with oncogenic properties overexpressed in PCa cases. Through the use of an alternative promoter, a truncated c-KIT protein (tr-KIT) of 30-50 kDa is generated, lacking the extracellular and transmembrane domain. Tr-KIT promotes the formation of a multi-molecular complex composed of Fyn, Plcγ1, and Sam68. Imatinib blocks the activity of full-length c-KIT but has no effect on tr-KIT. LNCaP is the human PCa cell line that shows tr-KIT overexpression and PC3 does not show tr-KIT overexpression. miR-128/193a- 5p/494 are miRNAs targeting FYN, PLCγ1, and SAM68 combinatorially. The study's question is: can miR-128/193a- 5p/494 be related to imatinib resistance in PCa? METHODS: LNCaP and PC3 cells were treated with imatinib in IC50 doses. Before and after imatinib administration, RNA was isolated and cDNA conversion was performed. By qPCR analysis, expression changes of tr-KIT specific pathway elements and miR-128/193a-5p/494 were analyzed before and after imatinib administration. RESULTS: After imatinib administration, miR-128/193a-5p/494 were significantly overexpressed in LNCaP cells while downregulated significantly in PC3 cells (p<0.05). Also, FYN was upregulated in LNCaP cells (p<0.05) but there was no change in PC3 after imatinib administration. CONCLUSION: Especially upregulation of FYN may sponge miR128/193a-5p/494 and downregulate their transcriptional activity in LNCaP cells having tr-KIT activity. So, miR-128/193a-5p/494 may have a critical role in imatinib resistance via a tr-KIT pathway.

Cytotoxic activities of Hypericum perforatum L. extracts against 2D and 3D cancer cell models.

Six extracts were obtained from plant species Hypericum perforatum L., collected at Samsun in Turkey. The aim of this study was to examine the mechanisms of the anticancer activity of these extracts. Methanol, ethyl-acetate and hexane were used as a solvents for extraction from both branch-body part of the plant (extracts 1, 2 and 3) and from plant flowers (extracts 4, 5 and 6). The cytotoxic effects of the extracts were determined against 2D and 3D cancer cell models. Cell cycle changes of treated HeLa cells were analyzed by flow cytometry. Measurements of gene and microRNA expression levels in treated HeLa cells were done by quantitative real time PCR. Five examined extracts (2-6) exerted selective concentration-dependent cytotoxic effects on HeLa, K562, and A549 cancer cells, while the extract 1 exhibited very weak cytotoxicity. The extract 6 showed the highest intensity of cytotoxic activity. All tested extracts (2-6) demonstrated the ability to induce apoptosis in HeLa cells through activation of caspase-3. These extracts remarkably decreased gene expression levels of MMP2, MMP9, TIMP3, and VEGFA in HeLa cells. Flower extracts might have stronger effects on miR128/193a-5p/335 level changes than branch-body extracts. Hypericum perforatum extracts exerted weaker cytotoxic effects on 3D HeLa spheroids when compared with their effects on 2D monolayer HeLa cells. Taken together, results of our research may suggest the promising anticancer properties of the Hypericum perforatum extracts.

Association of Abl interactor 2, ABI2, with platelet/lymphocyte ratio in patients with renal cell carcinoma: A pilot study.

There are many unknown aspects of the pathogenesis of renal cell carcinoma (RCC). The aim of the current study was to define new RCC-related genes and measure their associations with RCC and clinical parameters, especially platelet/lymphocyte ratio which may be an independent predictor of prognosis in patients with RCC and other forms of cancer. Via in silico analysis upon RCC-specific deleted genes in chromosome 3, four possible ceRNAs (ATXN3, ABI2, GOLGB1 and SMAD2) were identified. Then, the expression levels of these genes in tumour and adjacent healthy kidney tissues of 19 RCC patients were determined by real-time PCR. ATXN3 and GOLGB1 gene expression levels increased but ABI2 gene expression level decreased in tumour kidney tissues when compared to normal ones. ATXN3, ABI2 and GOLGB1 gene expression levels were significantly higher in Fuhrman grade 4 than other grades (P < .001). ABI2 gene expression levels were significantly associated with higher platelet/lymphocyte ratio of the patients with RCC (P < .05). ATXN3, ABI2 and GOLGB1 may predict higher RCC grades. Also, ABI2 may regulate platelet/lymphocyte ratio which may be an independent predictor of RCC and other forms of cancer.

Tr-KIT/c-KIT ratio in renal cell carcinoma.

Truncated KIT (tr-KIT) is an alternative variant of c-KIT protein. Previous studies have clearly documented that c-KIT was associated with various oncogenic processes in RCC. However, the biological significance of tr-KIT in RCC development and progression remains unclear. So, it was aimed to investigate the possible association between RCC and tr-KIT which is thought to activate some oncogenic pathways. In this study, Kidney Cancer cDNA Array containing a total of 48 cDNA samples from the normal kidney tissues of 9 healthy subjects and kidney tumor tissues of 10 stage-1, 5 stage-2, 13 stage-3 and 11 stage-4 RCC patients was used for gene expression analysis. Real-Time PCR method was used to measure tr-KIT/c-KIT expression ratios. tr-KIT/c-KIT expression ratio was compared between tumor and normal samples, and statistically correlated with the clinical parameters of RCC patients. tr-KIT/c-KIT expression ratio was approximately 4-times higher in tumor samples than control ones (p = 0.001). Also, tr-KIT/c-KIT expression ratio was approximately two, three and six times higher in Fuhrman nuclear grades 2, 3 and 4 than normal, respectively (p = 0.009). Moreover, clear cell and papillary RCC has a significantly higher level of tr-KIT/c-KIT expression ratio than chromophobe RCC (p = 0.016). In the current study, it was stated for the first time that tr-KIT/c-KIT expression ratio was up-regulated in RCC tissues, and high tr-KIT/c-KIT expression ratio was correlated with more aggressive clinical features and poor patient prognosis. Our results suggest that increased tr-KIT/c-KIT expression ratio might be useful as a prognostic marker for RCC patients.

Cross-Kingdom Gene regulation via miRNAs of Hypericum perforatum (St. John's wort) flower dietetically absorbed: An in silico approach to define potential biomarkers for prostate cancer.

Prostate cancer (PCa) is the most frequent type of cancer in men. Hypericum perforatum (H. Perforatum) extract (HPE) administration provides remarkable decrease of PCa development. H. perforatum contains 7 conserved miRNAs (Hyp-miR-156a, Hyp-miR-156b, Hyp-miR-166, Hyp-miR-390, Hyp-miR-394, Hyp-miR-396 and Hyp-miR-414) with different targets. In this study, we aimed to investigate cross-kingdom gene regulation via miRNAs of H. perforatum flower dietetically absorbed in manner of an in silico approach to define potential biomarkers for PCa. psRNATarget database was used to find human genes targeted by 7 pre-defined H. perforatum miRNAs. We defined the mostly affected gene families from these miRNAs as ZNF, TMEM, SLC and FAM gene families. GeneMANIA database was used to define the most affected genes (TMEM41B and SLC4A7) from these 7 miRNAs. cBioPortal database was used to define alteration frequencies of TMEM41B and SLC4A7 on different types of PCa and to measure the mutual interaction potency and significance of co-occurence in PCa. This analysis showed that neuroendocrine prostate cancer (NEPC) had the highest total mutation frequency (22%) of TMEM41B and SLC4A7 genes. Also, TMEM41B and SLC4A7 genes had an average 2.1% pathway change potential among all different types of PCa. Moreover, TMEM41B and SLC4A7 gene pair was found significantly co-occurrent in PCa (p < 0.001). Finally, via GEPIA database, we used Spearman correlation analysis to measure the correlation degree of TMEM41B and SLC4A7 genes in PCa and found their significant correlation with PCa (p = 1.2 × 10-12, R = 0.28). All in all, it was proved in silico and supported with previously known clinical data that SLC4A7 and TMEM41B potentially have a significant and critical tumor suppressive role for PCa, and show this effect combinatorily working together. This is the first study correlating SLC4A7 and TMEM41B with PCa significantly.

Glutamate transporter SLC1A1 is associated with clear cell renal cell carcinoma

Background/aim: This study aimed to comparatively analyze the expression levels of the SLC1A1 gene in renal specimens from tumors and adjacent healthy kidney tissues of patients with clear cell renal cell carcinoma (ccRCC). Materials and methods: Nineteen patients diagnosed with ccRCC were included in the study. The expression levels of the SLC1A1 and GAPDH genes were measured in tumor and formalin-fixed paraffin-embedded (FFPE) tissue specimens from the adjacent healthy kidney of each subject. Via the GEPIA database, the distribution of SLC1A1 gene expressions in ccRCC and healthy kidney tissues was obtained. The relative expression of SLC1A1 was evaluated for the association with the clinical parameters of the patients. Results: The expression of the SLC1A1 gene was significantly higher in males than females (P = 0.029). Also, there were statistically significant associations between stages II­IV and Fuhrman grades 2­4 with respect to SLC1A1 gene expression (P < 0.001 for both). Moreover, low levels of red blood cell and hemoglobin counts were significantly associated with the SLC1A1 expression (P < 0.001 and P = 0.005, respectively). The expression of the SLC1A1 gene in tumor tissues increased approximately 3 times compared with normal kidney tissues (P < 0.05). According to the GEPIA database, SLC1A1 gene expression is significantly higher in ccRCC patients than healthy persons (P = 0.01). Conclusion: The change in the expression of SLC1A1 may be crucial for ccRCC pathophysiology.

Significance of miR-15a-5p and CNKSR3 as Novel Prognostic Biomarkers in Non-Small Cell Lung Cancer.

BACKGROUND: In recent years, targeted cancer treatment methods at various molecular levels have been developed for Non-Small Cell Lung Cancer (NSCLC), one of two major subtypes of lung cancer. miRNAbased clinical trials are currently the preferred targeted therapeutic strategy. Also, ceRNAs (competing endogenous RNA) would be the newest and the most effective approach to uncover novel interactions between mRNAs and miRNAs in NSCLC carcinogenesis. There are many factors influencing the efficiency of a miRNA to suppress or silence translation of the target mRNA. The most effective event is the presence of other RNAs showing ceRNA activity. These RNAs contain binding sites for specific miRNAs and enable miRNAs to bind these pseudo targets, instead of the original binding sites on the target mRNA. Therefore, the mRNA of the target gene is less affected by this miRNA, while the amount of miRNA remains the same in the media. METHOD: For this project, we determined that five clinically important different oncogenes (PDL1, FGFR1, DDX3X, SLC1A5, FXR1 ) are involved in the pathogenesis of NSCLC. For this purpose, we transfected model NSCLC cell line, A549, with miRNAs (miR-150-5p, miR-15a-5p, miR-503-5p) targeting these oncogenes to investigate whether these oncogenes will be suppressed at the mRNA level and also how the suppression efficiency of these miRNA on the oncogenes will be affected by possible ceRNA (CNKSR3, POU2F1, HIPK2) activities. RESULTS: miR-15a-5p was determined to have the most suppressive effect on the five genes and three potential ceRNAs (p<0.05). Furthermore, CNKSR3 was the ceRNA most affected by all three miRNAs (p<0.05). CONCLUSION: CNKSR3 was affected more than the oncogenes known to act on NSCLC and this might make it a stronger and novel marker for use in possible treatment regimens designed using miR-15a-5p silencing effect on oncogenes in NSCLC pathogenesis. According to the literature, this is the first study associating NSCLC with miR-15a-5p and CNKSR3.

miRNAs as Potential Treatment Targets and Treatment Options in Cancer.

Standard cancer therapies for solid malignancies, such as chemotherapy and radiotherapy, are not target specific against cancer cells and are often not fully efficacious. Chemotherapy and radiotherapy may cause side effects, and the need to develop additional strategies for cancer treatment is urgent. MicroRNAs (miRNAs) are small non-coding RNAs with heterogeneous functions and have been described in almost every known cancer model. Besides their basic tumor-suppressive and oncogenic functions, they also have the potential to modulate chemotherapy and radiotherapy and to be manipulated with chemical compounds to make them chemically suitable for efficient delivery to cancer cells. It has been suggested that the level of expression of specific miRNAs could increase treatment efficacy by determining the stage of chemotherapy/radiotherapy sensitivity. Application of miRNAs alone or in combination with standard therapeutic strategies may significantly improve the success of cancer treatments in the future.

Levels of MicroRNA Heterogeneity in Cancer Biology.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression, involved in the silencing of messenger RNA (mRNA) translation. The importance of miRNA signatures in disease screening, prognosis, and progression of different tumor types and subtypes is increasing. miRNA expression levels change depending on numerous factors. In this review, we are describing the circumstances under which miRNA levels can change, these are named 'levels' of heterogeneity of miRNAs. miRNAs can have oncogenic, tumor suppressive, or both roles depending on tumor type and target mRNA whose translation they silence. The expression levels of a single miRNA may vary across different cancer types and subtypes, indicating that a miRNA signature may be tissue specific. miRNA levels of expression also vary during disease formation and propagation, indicating the presence of a time profile for their expression. The complexity of the miRNA-mRNA interference network mirrors different genetic and epigenetic changes that influence miRNA and mRNA availability to each other, and hence, their binding ability. The potential role of miRNAs as biomarkers is two-fold; first, for monitoring of the phases of cancer pathogenesis, and second, to characterize the particular type/subtype of cancer. It is important that a particular miRNA should be characterized by examining as many types and subtypes of cancers as are available, as well as being extracted from different types of samples, in order to obtain a complete picture of its behavior and importance in the disease pathology.

Sequence-based analysis of 5'UTR and coding regions of CASP3 in terms of miRSNPs and SNPs in targetting miRNAs.

Apoptosis is described as a mechanism of cell death occurring after adequate cellular harm. Deregulation of apoptosis occurs in many human conditions such as autoimmune disorders, ischemic damage, neurodegenerative diseases and different cancer types. Information relating miRNAs to cancer is increasing. miRNAs can affect development of cancer via many different pathways, including apoptosis. Polymorphisms in miRNA genes or miRNA target sites (miRSNPs) can change miRNA activity. Although polymorphisms in miRNA genes are very uncommon, SNPs in miRNA-binding sites of target genes are quite common. Many researches have revealed that SNPs in miRNA target sites improve or decrease the efficacy of the interaction between miRNAs and their target genes. Our aim was to specify miRSNPs on CASP3 gene (caspase-3) and SNPs in miRNA genes targeting 5'UTR and coding exons of CASP3, and evaluate the effect of these miRSNPs and SNPs of miRNA genes with respect to apoptosis. We detected 141 different miRNA binding sites (126 different miRNAs) and 7 different SNPs in binding sites of miRNA in 5'UTR and CDS of CASP3 gene. Intriguingly, miR-339-3p's binding site on CASP3 has a SNP (rs35372903, G/A) on CASP3 5'UTR and its genomic sequence has a SNP (rs565188493, G/A) at the same nucleotide with rs35372903. Also, miR-339-3p has two other SNPs (rs373011663, C/T rs72631820, A/G) of which the first is positioned at the binding site. Here, miRSNP (rs35372903) at CASP3 5'UTR and SNP (rs565188493) at miR-339-3p genomic sequence cross-matches at the same site of binding region. Besides, miR-339-3p targets many apoptosis related genes (ZNF346, TAOK2, PIM2, HIP1, BBC3, TNFRSF25, CLCF1, IHPK2, NOL3) although it had no apoptosis related interaction proven before. This means that miR-339-3p may also have a critical effect on apoptosis via different pathways other than caspase-3. Hence, we can deduce that this is the first study demonstrating a powerful association between miR-339-3p and apoptosis upon computational analysis.

Oncocers: ceRNA-mediated cross-talk by sponging miRNAs in oncogenic pathways.

Competing endogenous RNAs (ceRNAs) are RNA transcripts which can communicate with each other by decreasing targeting concentration of micro-RNA (miRNA) with the derepression of other messenger RNAs (mRNAs) having the common miRNA response elements (MREs). Oncocers are ceRNAs taking crucial roles in oncogenic pathways processed in many types of cancer, and this study analyzes oncocer-mediated cross-talk by sponging microRNAs (miRNAs) in these pathways. While doing this, breast, liver, colon, prostate, gastric, lung, endometrium, thyroid and epithelial cancers and melanoma, rhabdomyosarcoma, glioblastoma, acute promyelocytic leukemia, retinoblastoma, and neuroblastoma were analyzed with respect to ceRNA-based carcinogenesis. This study defines, firstly, oncocers in the literature and contains all oncocer-related findings found up to now. Therefore, it will help to increase our comprehension about oncocer-mediated mechanisms. Via this study, a novel perspective would be produced to make clear cancer mechanisms and suggest novel approaches to regulate ceRNA networks via miRNA competition for cancer therapeutics. Graphical Abstract Multiple RNA transcripts have common MREs for the similar miRNA in their 3'-untranslated regions (3'-UTRs). Upregulation of ceRNAs rises the abundance of specific MREs and shifts the miRNA pool distribution, as a result, leading to the increased expression of target mRNA. The depot of genomic mutations and epigenetic alterations changing gene function and expression causes cancers. Herewith, genome-based somatic base-pair mutations, DNA copy number alterations, chromosomal translocation, also transcript fusions, alternative splicing are usually seen in cancer situations. Consequently, such cases causing changed UTR expression in transcripts influence the levels of MRE or present new MREs into the cells. Alterations in MREs of ceRNAs affect the capability of a specific mRNA transcript to attach or titrate miRNAs. As a result, the disturbed ceRNA network can lead to diseases and cancers. As a new term in RNA world, oncocers-the name for ceRNAs taking crucial roles in oncogenic pathways-are processed in many types of cancer, and oncocer-mediated cross-talk are analyzed by sponging miRNAs in these pathways.

Assessment of the effect of laser irradiations at different wavelengths (660, 810, 980, and 1064 nm) on autophagy in a rat model of mucositis.

It is known that high-dose radiation has an effect on tissue healing, but tissue healing does not occur when low dose radiation is applied. To clarify this issue, we compare the treatment success of low dose radiation with programmed cell death mechanisms on wounded tissue. In this study, we aimed to investigate the interactions of low and high-dose radiation using an autophagic mechanism. We included 35 adult Wistar-Albino rats in this study. All animals were injected with 100 mg/kg of 5-fluorouracil (5-FU) on the first day and 65 mg/kg of 5-FU on the third day. The tips of 18-gauge needles were used to develop a superficial scratching on the left cheek pouch mucosa by dragging in a linear movement on third and fifth days. After mucositis formation was clinically detected, animals were divided into five groups (n = 7). Different wavelengths of laser irradiations (1064 nm, Fidelis Plus, Fotona, Slovenia; 980 nm, FOX laser, A.R.C., Germany; 810 nm, Fotona XD, Fotona, Slovenia; 660 nm, HELBO, Medizintechnik GmbH, Wels, Austria) were performed on four groups once daily for 4 days. The laser irradiation was not performed on the control group. To get the tissue from the left cheek at the end of fourth day from all animals, oval excisional biopsy was performed. Molecular analysis assessments of pathological and normal tissue taken were performed. For this purpose, the expression analysis of autophagy genes was performed. The results were evaluated by normalization and statistics analysis. We found that Ulk1, Beclin1, and Atg5 expression levels were increased in the rats when the Nd:YAG laser was applied. This increase showed that a 1064-nm laser is needed to activate the autophagic mechanism. However, in the diode applications, we found that Beclin1, Atg10, Atg5, and Atg7 expressions numerically decreased. Atg5 is responsible for the elongation of autophagosome. Becn1 is a control gene in the control mechanism of autophagy. The reduction of the expression of these genes leads us to think that it may depend on the effect of drug (5-FU) used to form model. Expressions of therapeutic genes increase to ensure hemostasis, but in our study, expressions were found to decrease. More detailed studies are needed.

The association of the expression of miR-122-5p and its target ADAM10 with human breast cancer.

MicroRNAs can regulate many biological functions. miR-122-5p has a tumor suppressor function through different molecular pathways. Also, our second hit, ADAM10, targeted by miR-122-5p, is a major determinant of HER2 shedding causing that trastuzumab cannot bind to HER2 receptors. Therefore, our analysis upon ADAM10 expression and miR-122-5p was a good point to understand molecular mechanism of breast cancer. In our study, we investigated the expression profiles of miR-122-5p and its target ADAM10 in 71 breast cancer patients. Immunohistochemical analysis of ER, PR and HER2 gene products was used to categorize tumors in patients. Expression data and immunohistochemical findings were evaluated to comment on the relationship between miR-122-5p and ADAM10. ADAM10 expression was higher in tumor than that of normal tissue but miR-122-5p expression was lower in tumor than that of normal tissue. The expression pattern in HER2+ patients was reverse of the overall result. It can be explained like that miR-122-5p expression increases especially in HER2+ cancer cell to suppress ADAM10 shedding activity on HER2 receptor. However, increase in expression of tumor suppressor miR-122-5p is not enough to inhibit ADAM10. All in all, we can think miR-122-5p as potential regulator of ADAM10 and trastuzumab resistance. Since if we increase miR-122-5p activity together with trastuzumab administration, then HER2+ breast cancer cells may overcome trastuzumab resistance by inhibiting ADAM10 shedding activity on HER2 receptors and increase the efficiency of trastuzumab.

The investigation of miR-221-3p and PAK1 gene expressions in breast cancer cell lines.

The most common malignancy in women is breast cancer. Drug resistance in the treatment of cancer still remains a major clinical concern. Resistance to tamoxifen is seen in half of the recurrences in breast cancer. The anti-estrogen tamoxifen gains agonistic property by transactivating ERα. PAK1-mediated phosphorylation of serine 305 (S305) of ERα leads to resistance to tamoxifen. In our study, PAK1-induced suggestive tamoxifen resistance was designed. According to our hypothesis, phosphorylation of ERα-S305 by PAK1 may be reversed by PAK1 transcriptional inhibition by miR-221-3p due to miR-221-3p targeting the 3' UTR of PAK1. For this purpose, we used Real-time PCR (qRT-PCR) to measure the expression level of miR-221-3p in ER-positive breast cancer cell lines (ZR-75-1, MCF7) and breast epithelial cell line, hTERT-HME1, as control in the laboratory in our department. The increase in the expression of PAK1 depending on miR-221-3p may be related to ZR-75-1 cell line which has invasive characteristic but other two ER+ cancer cell lines, MCF7 and HCC1500, have milder cancer severity. miR-221-3p may have a role on regulation of PAK1 expression because miR-221-3p expression level decreases while PAK1 expression level increases in SKBR3 cell line. miR-221-3p and PAK1 expressions in MDA-MB-231 cell line are higher than that of hTERT-HME1 cell line. This may mean that miR-221-3p has no regulatory effect on of PAK1 expression in this cell line. According to these results, miR-221-3p may give crucial information about molecular mechanism of the disease upon PAK1 activity or different mechanisms with respect to histopathology and severity of breast cancer.

Computational analysis of 3'UTR region of CASP3 with respect to miRSNPs and SNPs in targetting miRNAs.

Apoptosis is a strictly organized course which keeps the healthy survival/death equilibrium. Disregulation in apoptosis may lead autoimmunity or cancer, but increased apoptosis can lead degenerative diseases. Studies during the last several years have identified numerous affected miRNAs in association with apoptosis, their target genes and biological functions, and possible drug interventions. Polymorphisms in miRNA genes or miRNA target sites (miRSNPs) can modify miRNA action. While polymorphisms in miRNA genes are relatively rare, SNPs in miRNA-binding sites in target genes are more frequent. Several studies have shown that SNPs in miRNA target sites enhance or weaken the interaction between miRNA and its target transcripts and are associated with cancers and other diseases. We aimed to identify miRSNPs on executioner caspase, CASP3 gene (caspase-3) and SNPs in miRNA genes targeting 3'UTR of CASP3 and assessing the impact of these miRSNPs and SNPs of miRNA genes targeting 3'UTR of CASP3 with respect to apoptosis. We identified 89 different miRNA binding sites (for 43 different miRNAs) and 16 different SNPs in binding sites of miRNA in the 3'UTR of the CASP3 gene. Also, 2 SNPs (rs372435266 and rs190144655) were found on this miRNA' genomic sequence. One of them crossmatched with a SNP in the 3'UTR of CASP3 that we found formerly. This miRNA was miR-4802-3p. Besides, miR-4802-3p targets three other apoptosis related genes, XIAP, IL1A and SOX2. This means that miR-4802-3p may also have a critical effect on apoptosis via different pathways other than caspase-3. We can therefore conclude that this is the first study proving a strong association between miR-4802-3p and apoptosis upon computational targetting analysis.

miR-204-5p expression in colorectal cancer: an autophagy-associated gene.

MicroRNAs (miRNAs) are important factors during tumorigenesis by affecting posttranscriptional gene expression. miRNA 204 (miR-204) is a miRNA frequently investigated in different types of cancers. According to literature, autophagy has dual roles in cancer, acting as both a tumor suppressor and cell survival agent. Also, the current data suggests that autophagy is activated in human colorectal cancer cells and enhances the aggressiveness of human colorectal cancer cells. So, our aim is to investigate potential effect of miR-204-5p on colorectal cancer by associating its expression with autophagy-related targets of miR-204-5p. This is the first miRNA study conducted on patients with colorectal cancer and healthy subjects and also to search the relation of miR-204-5p with clinicopathological factors and survival. Sixty-six patients with colorectal cancer and healthy subjects without any known chronic disease were enrolled into our study. Total miRNA was isolated from paraffin-embedded tissues of all patients' cancerous and normal tissues, and healthy subjects. cDNAs were obtained from this miRNAs by reverse transcriptase method, and miR-204-5p relative expression levels were detected by qRT-PCR method. Patients were divided into two groups according to median relative expression levels of miR-204-5p, as low- and high-expression group. Relation of miR-204-5p with clinicopathological factors and overall survival was also investigated. Medians of miR-204-5p relative expression levels in cancerous and normal tissues of patients were found as 0.00235 and 0.00376, respectively. The difference between two groups was not statistically significant (p = 0.11). Nonetheless, median of miR-204-5p relative expression levels in healthy subjects were found as 0.00135, and the difference between patient with cancer and healthy subjects and between normal tissues of patients and healthy subjects were statistically significant (p = 0.021 and p = 0.0005, respectively). There were 32 patients (48.5 %) showing high expression and 34 patients (51.5 %) showing low expression according to miR-204-5p relative expression levels. There were no statistically significant relation between clinicopathologic features and miR-204-5p relative expression levels. We also investigated the relation between miR-204-5p relative expression levels and overall survival, and no statistically significant relation was found between them (p = 0.462). The absence of any significant difference between tumor and non-tumor samples, low sample size, and performance at just one center are the limitations of our study. In opposition to literature, miR-204-5p is overexpressed in colorectal cancer patients as compared with healthy subjects and this situation is not associated with clinicopathological factors and overall survival. This may be explained by the fact that miR-204-5p increases in colorectal cancer cases in order to inhibit increased activity of LC3B-II in autophagy and Bcl2 against apoptosis posttranscriptionally and to take role as tumor suppressor.

The interrelationship between HER2 and CASP3/8 with apoptosis in different cancer cell lines.

HER2/ErbB2, a known proto-oncogene (also known as HER2, neu), is among the most practiced molecules in the cancer area. Human epidermal growth factor receptor 2 (HER2) is over expressed in approximately 20-30 % of breast cancer tumors and also in a lot of other human cancer types. It is known to be related to the aggressiveness of the disease, increased mortality and higher relapse ratio. The unusual HER2 overexpression is associated with more severe disease characteristics in several cancers. In recent past, there have been remarkable advances in understanding the role of the HER2 gene in cancers. Caspases are well renowned proteases that act as essential initiators and executioners of the apoptotic process. The primary function of HER2 is suppressing apoptosis to enhance cell survival and eventually giving rise to uncontrolled proliferation and tumor growth. The objective of this work was to study the expression levels of HER2 and apoptosis related factors CASP-3 and CASP-8 in several breast and other cancer cell lines and finally to find a meaningful correlation between all these. We summed up by obtaining an increase in expression of HER2 in all cancer cell lines as compared to that of CASP-3 and CASP-8. In summary we conclude that HER2 promotes cell survival by inhibiting apoptosis i.e. by downregulating CASP-3 and CASP-8. This is a novel study comprising the expression study of HER2 and different caspases in different cancer cell lines simultaneously. It is thus expected that this study will aid in better establishment of correlation between HER2 and caspases in different malignancies.

Expression patterns of miR-221 and its target Caspase-3 in different cancer cell lines.

Caspases are important initiators and most well-known finishers of apoptosis. By changing the death propagation homeostatic equilibrium, their different expression patterns might trigger the progression of hazardous diseases like cancer. miR-221 is an oncogenic miRNA. It is known to have both anti-angiogenic and angiogenic effect. The aim of this work was to compare the expression levels of miR-221 and its target caspase-3 in different cancer cell lines and to find out a relationship between these two. We also tried to establish a prominent relationship between miR-221 and its role in apoptosis by studying their expression levels. Our results indicate that expression of caspase-3 is quite lower as compared to miR-221 expression in all of the selected cancer cell lines. As a result, we conclude that miR-221 may have a crucial role in repressing the expression of caspase-3 which may contribute to a lower apoptotic rate, thus supporting the selection of more aggressive cancer cells. To our knowledge, this is the first study related to the expression levels of caspase-3 and miR-221 in different cell lines at the same time. We expect that our study might pave the way for better understanding the role of miR-221 in apoptotic regulation of caspase-3.