Role of the 3-mercaptopyruvate sulfurtransferase in colon/colorectal cancers.

The 3-mercaptopyruvate sulfurtransferase (MPST) is a protein persulfidase, occurring mainly in mitochondria. Although function of this protein in cancer cells has been already studied, no clear outcome can be postulated up to now. Therefore, we focused on the determination of function of MPST in colon (HCT116 cells)/colorectal (DLD1 cells) cancers. In silico analysis revealed that in gastrointestinal cancers, MPST together with its binding partners can be either of a high risk or might have a protective effect. Silencing of MPST gene resulted in decreased ATP, while acetyl-CoA levels were elevated. Increased apoptosis was detected in cells with silenced MPST gene, which was accompanied by decrease in mitochondrial membrane potential, but no changes in IP3 receptor's protein. Mitochondria underwent activation of fission and elevated DRP1 expression after MPST silencing. Proliferation and migration of DLD1 and HCT116 cells were markedly affected, showing the importance of MPST protein in colon/colorectal cancer development.

Biallelic Cys141Tyr variant of SEL1L is associated with neurodevelopmental disorders, agammaglobulinemia, and premature death.

Suppressor of lin-12-like-HMG-CoA reductase degradation 1 (SEL1L-HRD1) ER-associated degradation (ERAD) plays a critical role in many physiological processes in mice, including immunity, water homeostasis, and energy metabolism; however, its relevance and importance in humans remain unclear, as no disease variant has been identified. Here, we report a biallelic SEL1L variant (p. Cys141Tyr) in 5 patients from a consanguineous Slovakian family. These patients presented with not only ERAD-associated neurodevelopmental disorders with onset in infancy (ENDI) syndromes, but infantile-onset agammaglobulinemia with no mature B cells, resulting in frequent infections and early death. This variant disrupted the formation of a disulfide bond in the luminal fibronectin II domain of SEL1L, largely abolishing the function of the SEL1L-HRD1 ERAD complex in part via proteasomal-mediated self destruction by HRD1. This study reports a disease entity termed ENDI-agammaglobulinemia (ENDI-A) syndrome and establishes an inverse correlation between SEL1L-HRD1 ERAD functionality and disease severity in humans.

Prognostic Value of Genotype-Phenotype Correlations in X-Linked Myotubular Myopathy and the Use of the Face2Gene Application as an Effective Non-Invasive Diagnostic Tool.

BACKGROUND: X-linked myotubular myopathy (XLMTM) is a rare congenital myopathy resulting from dysfunction of the protein myotubularin encoded by the MTM1 gene. XLMTM has a high neonatal and infantile mortality rate due to a severe myopathic phenotype and respiratory failure. However, in a minority of XLMTM cases, patients present with milder phenotypes and achieve ambulation and adulthood. Notable facial dysmorphia is also present. METHODS: We investigated the genotype-phenotype correlations in newly diagnosed XLMTM patients in a patients' cohort (previously published data plus three novel variants, n = 414). Based on the facial gestalt difference between XLMTM patients and unaffected controls, we investigated the use of the Face2Gene application. RESULTS: Significant associations between severe phenotype and truncating variants (p < 0.001), frameshift variants (p < 0.001), nonsense variants (p = 0.006), and in/del variants (p = 0.036) were present. Missense variants were significantly associated with the mild and moderate phenotype (p < 0.001). The Face2Gene application showed a significant difference between XLMTM patients and unaffected controls (p = 0.001). CONCLUSIONS: Using genotype-phenotype correlations could predict the disease course in most XLMTM patients, but still with limitations. The Face2Gene application seems to be a practical, non-invasive diagnostic approach in XLMTM using the correct algorithm.

Severe congenital T-lymphocytopenia may affect the outcome of neonatal intensive care.

AIM: Circular DNA segments TREC (T-cell receptor excision circles) formed during T-lymphocyte maturation in the thymus, are a sensitive marker of thymic lymphocyte production in a broader manner. Quantification using qPCR is proposed as a surrogate marker of T cell malfunction in various primary and secondary conditions in a non-SCID selected risk newborn population. METHODS: We collected 207 dry blood spot samples during the years 2015-2018, from newly admitted risk newborns. TREC values calculated per 106 cells were determined and a cut-off values of 5th percentile was set. The positive control group consisted of patients (n=13) with genetically confirmed SCID. RESULTS: The median TREC value was 34,591.56 (18,074.08-60,228.58) for girls resp. 28,391.20 (13,835.01-51,835.93) per 106 cells for boys, P=0.046. Neonates born by C-section have been found to have higher TREC levels compared to neonates born by spontaneous delivery (P=0.018). In the group of preterm newborns (n=104), 3.8% had TREC value < 5th percentile, half of them died due to sepsis as opposed to no fatalities in preterm newborns with sepsis and TREC value > 5th percentile. In the group of term newborns (n=103) 9 children (8.7%) had TREC < 5th percentile, half of them were treated for asphyxia, with no fatal complications. CONCLUSION: TREC levels calculated for the 5th percentile of a risk neonatal group is suggested as a surrogate marker for increased risk of fatal septic complication. Early recognition of these newborns within a risk scoring system using TREC levels could lead to potentially lifesaving interventions.

Genome-wide DNA methylome and transcriptome changes induced by inorganic nanoparticles in human kidney cells after chronic exposure.

The unique physicochemical properties make inorganic nanoparticles (INPs) an exciting tool in diagnosis and disease management. However, as INPs are relatively difficult to fully degrade and excrete, their unintended accumulation in the tissue might result in adverse health effects. Herein, we provide a methylome-transcriptome framework for chronic effects of INPs, commonly used in biomedical applications, in human kidney TH-1 cells. Renal clearance is one of the most important routes of nanoparticle excretion; therefore, a detailed evaluation of nanoparticle-mediated nephrotoxicity is an important task. Integrated analysis of methylome and transcriptome changes induced by INPs (PEG-AuNPs, Fe3O4NPs, SiO2NPs, and TiO2NPs) revealed significantly deregulated genes with functional classification in immune response, DNA damage, and cancer-related pathways. Although most deregulated genes were unique to individual INPs, a relatively high proportion of them encoded the transcription factors. Interestingly, FOS hypermethylation inversely correlating with gene expression was associated with all INPs exposures. Our study emphasizes the need for a more comprehensive investigation of INPs' biological safety, especially after chronic exposure.

Breakpoints characterisation of the genomic deletions identified by MLPA in alkaptonuria patients.

Until recently, mainly DNA sequencing has been used to identify variants within the gene coding for homogentisate dioxygenase (HGD, 3q13.33) that cause alkaptonuria (AKU), an autosomal recessive inborn error of metabolism of tyrosine. In order to identify possible larger genomic deletions we have developed a novel Multiplex Ligation-dependent Probe Amplification (MLPA) assay specific for this gene (HGD-MLPA) and tested it successfully in healthy controls and in patients carrying two known previously identified HGD deletions. Subsequently, we analysed 22 AKU patients in whom only one or none classical HGD variant was found by sequencing. Using HGD-MLPA and sequencing, we identified four larger deletions encompassing from 1 to 4 exons of this gene and we defined their exact breakpoints: deletion of exons 1-4 (c.1-8460_282 + 6727del), deletion of exons 5 and 6 (c.283-9199_434 + 1688del), deletion of exon 11 (c.775-1915_879 + 1293del), and deletion of exon 13 (c.1007-1709_1188 + 1121del). We suggest including MLPA in the DNA diagnostic protocols for AKU in cases where DNA sequencing does not lead to identification of both HGD variants.

Decitabine-induced DNA methylation-mediated transcriptomic reprogramming in human breast cancer cell lines; the impact of DCK overexpression.

Decitabine (DAC), a DNA methyltransferase (DNMT) inhibitor, is tested in combination with conventional anticancer drugs as a treatment option for various solid tumors. Although epigenome modulation provides a promising avenue in treating resistant cancer types, more studies are required to evaluate its safety and ability to normalize the aberrant transcriptional profiles. As deoxycytidine kinase (DCK)-mediated phosphorylation is a rate-limiting step in DAC metabolic activation, we hypothesized that its intracellular overexpression could potentiate DAC's effect on cell methylome and thus increase its therapeutic efficacy. Therefore, two breast cancer cell lines, JIMT-1 and T-47D, differing in their molecular characteristics, were transfected with a DCK expression vector and exposed to low-dose DAC (approximately IC20). Although transfection resulted in a significant DCK expression increase, further enhanced by DAC exposure, no transfection-induced changes were found at the global DNA methylation level or in cell viability. In parallel, an integrative approach was applied to decipher DAC-induced, methylation-mediated, transcriptomic reprogramming. Besides large-scale hypomethylation, accompanied by up-regulation of gene expression across the entire genome, DAC also induced hypermethylation and down-regulation of numerous genes in both cell lines. Interestingly, TET1 and TET2 expression halved in JIMT-1 cells after DAC exposure, while DNMTs' changes were not significant. The protein digestion and absorption pathway, containing numerous collagen and solute carrier genes, ranking second among membrane transport proteins, was the top enriched pathway in both cell lines when hypomethylated and up-regulated genes were considered. Moreover, the calcium signaling pathway, playing a significant role in drug resistance, was among the top enriched in JIMT-1 cells. Although low-dose DAC demonstrated its ability to normalize the expression of tumor suppressors, several oncogenes were also up-regulated, a finding, that supports previously raised concerns regarding its broad reprogramming potential. Importantly, our research provides evidence about the involvement of active demethylation in DAC-mediated transcriptional reprogramming.

The importance of defining the age-specific TREC/KREC levels for detection of various inborn errors of immunity in pediatric and adult patients.

This study aimed to establish physiological TREC/KREC values in a healthy population of different ages to create cut-offs and analyze pediatric patients with various inborn errors of immunity. Dry blood spots and DNA samples purified from whole blood were used for TREC/KREC quantification using real-time PCR. Observed difference (p < 0.001) between methods revealed the isolation method as a factor we need to consider when determinating cut-offs. Data of 713 healthy individuals showed a negative correlation (p < 0.001) between age and TREC/KREC levels with gender difference observed only for KREC in a group of 51-60 years old (p < 0.001). The 5th percentile cut-off values were set for age groups, which allowed us to identify 25% of patients with immunodeficiencies in case of non-zero, borderline values of TREC/KREC. Screening of infants with congenital heart disease identified 11% of patients with lowered TREC/KREC and shows potential for newborn screening of specific groups of patients.

Alkaptonuria in Russia.

Alkaptonuria is characterized by the accumulation of homogentisic acid (HGA), part of which is excreted in the urine but the excess HGA forms a dark brown ochronotic pigment that deposits in the connective tissue (ochronosis), eventually leading to early-onset severe arthropathy. We analyzed a cohort of 48 Russian AKU families by sequencing all 14 exons (including flanking intronic sequences) of the homogentisate 1,2-dioxygenase gene (HGD) and Multiplex Ligation-dependent Probe Amplification (MLPA) analysis. Nine novel likely pathogenic HGD variants were identified, which have not been reported previously in any other country. Recently, Bychkov et al. [1] reported on the variant spectrum in another cohort of 49 Russian AKU patients. Here we summarize complete data from both cohorts that include 82 Russian AKU families. Taken together, 31 different HGD variants were found in these patients, of which 14 are novel and found only in Russia. The most common variant was c.481G>A (p.(Gly161Arg)), present in almost 54% of all AKU alleles.

KIT Expression Is Regulated by DNA Methylation in Uveal Melanoma Tumors.

Uveal melanoma (UM) is an ocular tumor with a dismal prognosis. Despite the availability of precise molecular and cytogenetic techniques, clinicopathologic features with limited accuracy are widely used to predict metastatic potential. In 51 UM tissues, we assessed a correlation between the expression of nine proteins evaluated by immunohistochemistry (IHC) (Melan-A, S100, HMB45, Cyclin D1, Ki-67, p53, KIT, BCL2, and AIFM1) and the presence of UM-specific chromosomal rearrangements measured by multiplex ligation-dependent probe amplification (MLPA), to find IHC markers with increased prognostic information. Furthermore, mRNA expression and DNA methylation values were extracted from the whole-genome data, achieved by analyzing 22 fresh frozen UM tissues. KIT positivity was associated with monosomy 3, increasing the risk of poor prognosis more than 17-fold (95% CI 1.53-198.69, p = 0.021). A strong negative correlation was identified between mRNA expression and DNA methylation values for 12 of 20 analyzed positions, five located in regulatory regions of the KIT gene (r = -0.658, p = 0.001; r = -0.662, p = 0.001; r = -0.816; p < 0.001; r = -0.689, p = 0.001; r = -0.809, p < 0.001, respectively). DNA methylation ß values were also inversely associated with KIT protein expression (p = 0.001; p = 0.001; p = 0.015; p = 0.025; p = 0.002). Our findings, showing epigenetic deregulation of KIT expression, may contribute to understanding the past failure to therapeutically target KIT in UM.

Mathematical modeling based on RT-qPCR analysis of SARS-CoV-2 in wastewater as a tool for epidemiology.

Coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerges to scientific research and monitoring of wastewaters to predict the spread of the virus in the community. Our study investigated the COVID-19 disease in Bratislava, based on wastewater monitoring from September 2020 until March 2021. Samples were analyzed from two wastewater treatment plants of the city with reaching 0.6 million monitored inhabitants. Obtained results from the wastewater analysis suggest significant statistical dependence. High correlations between the number of viral particles in wastewater and the number of reported positive nasopharyngeal RT-qPCR tests of infected individuals with a time lag of 2 weeks/12 days (R2 = 83.78%/R2 = 52.65%) as well as with a reported number of death cases with a time lag of 4 weeks/27 days (R2 = 83.21%/R2 = 61.89%) was observed. The obtained results and subsequent mathematical modeling will serve in the future as an early warning system for the occurrence of a local site of infection and, at the same time, predict the load on the health system up to two weeks in advance.

Clinical manifestation of CDKL5 deficiency disorder and identified mutations in a cohort of Slovak patients.

CDKL5 deficiency disorder (CDD) is an independent clinical entity associated with early-onset encephalopathy, which is often considered the type of epileptic encephalopathy with CDKL5 mutation also found in children diagnosed with early-onset seizure (Hanefeld) type of Rett syndrome, epileptic spasms, West syndrome, Lennox-Gastaut syndrome, or autism. Since early seizure onset is a prominent feature, in this study, a cohort of 54 unrelated patients consisting of 26 males and 28 females was selected for CDKL5 screening, with seizures presented before 12 months of age being the only clinical criterion. Five patients were found to have pathogenic or likely pathogenic variants in CDKL5 while 1 was found to have a variant of uncertain significance (p.L522V). Although CDKL5 variants are more frequently identified in female patients, we identified three male and three female patients (11.1 %, 6/54) in this study. Missense variant with unknown inheritance (p.L522V), de novo missense variant (p.E60 K), two de novo splicing (IVS15 + 1G > A, IVS16 + 2 T > A), and one de novo nonsense variant p.W125* were identified using Sanger sequencing. Whole exome analysis approach revealed de novo frameshift variant c.1247_1248delAG in a mosaic form in one of the males. Patient clinical features are reviewed and compared to those previously described in related literature. Variable clinical features were presented in CDKL5 positive patients characterised in this study. In addition to more common features, such as early epileptic seizures, severe intellectual disability, and gross motor impairment, inappropriate laughing/screaming spells and hypotonia appeared at the age of 1 year in all patients, regardless of the type of CDKL5 mutation or sex. All three CDKL5 positive males from our cohort were initially diagnosed with West syndrome, which suggests that the CDKL5 gene mutations are a significant cause of West syndrome phenotype, and also indicate the overlapping characteristics of these two clinical entities.

Monosomy 3 Influences Epithelial-Mesenchymal Transition Gene Expression in Uveal Melanoma Patients; Consequences for Liquid Biopsy.

Despite outstanding advances in diagnosis and the treatment of primary uveal melanoma (UM), nearly 50% of UM patients develop metastases via hematogenous dissemination, driven by the epithelial-mesenchymal transition (EMT). Despite the failure in UM to date, a liquid biopsy may offer a feasible non-invasive approach for monitoring metastatic disease progression and addressing protracted dormancy. To detect circulating tumor cells (CTCs) in UM patients, we evaluated the mRNA expression of EMT-associated transcription factors in CD45-depleted blood fraction, using qRT-PCR. ddPCR was employed to assess UM-specific GNA11, GNAQ, PLCß4, and CYSLTR2 mutations in plasma DNA. Moreover, microarray analysis was performed on total RNA isolated from tumor tissues to estimate the prognostic value of EMT-associated gene expression. In total, 42 primary UM and 11 metastatic patients were enrolled. All CD45-depleted samples were negative for CTC when compared to the peripheral blood fraction of 60 healthy controls. Tumor-specific mutations were detected in the plasma of 21.4% patients, merely, in 9.4% of primary UM, while 54.5% in metastatic patients. Unsupervised hierarchical clustering of differentially expressed EMT genes showed significant differences between monosomy 3 and disomy 3 tumors. Newly identified genes can serve as non-invasive prognostic biomarkers that can support therapeutic decisions.

Variant c.2158-2A>G in MANBA is an important and frequent cause of hereditary hearing loss and beta-mannosidosis among the Czech and Slovak Roma population- evidence for a new ethnic-specific variant.

BACKGROUND: The Roma are a European ethnic minority threatened by several recessive diseases. Variants in MANBA cause a rare lysosomal storage disorder named beta-mannosidosis whose clinical manifestation includes deafness and mental retardation. Since 1986, only 23 patients with beta-mannosidosis and biallelic MANBA variants have been described worldwide. RESULTS: We now report on further 10 beta-mannosidosis patients of Roma origin from eight families in the Czech and Slovak Republics with hearing loss, mental retardation and homozygous pathogenic variants in MANBA. MANBA variant c.2158-2A>G screening among 345 anonymized normal hearing controls from Roma populations revealed a carrier/heterozygote frequency of 3.77%. This is about 925 times higher than the frequency of this variant in the gnomAD public database and classifies the c.2158-2A>G variant as a prevalent, ethnic-specific variant causing hearing loss and mental retardation in a homozygous state. The frequency of heterozygotes/carriers is similar to another pathogenic variant c.71G>A (p.W24*) in GJB2, regarded as the most frequent variant causing deafness in Roma populations. CONLCUSION: Beta-mannosidosis, due to a homozygous c.2158-2A>G MANBA variant, is an important and previously unknown cause of hearing loss and mental retardation among Central European Roma.

Studying the Gene Expression of Penicillium rubens Under the Effect of Eight Essential Oils.

Essential oils (EOs) are well-known for their beneficial properties against a broad range of microorganisms. For the better understanding of their mechanism of action in fungi, a microarray approach was used in order to evaluate the gene expression of Penicillium chrysogenum (recently renamed P. rubens) exposed to the indirect contact (vapors) of eight EOs. The selection of assayed EOs was based on their antifungal activity. The extraction of RNA and the microarray hybridization procedure were optimized for the analysis of P. rubens. Gene ontology annotation was performed to investigate the functional analysis of the genes. To uncover the metabolic pathway of these differentially expressed genes, they were mapped into the KEGG BRITE pathway database. The transcriptomic analysis showed that, from a total of 12,675 genes, only 551 genes are annotated, and the other 12,124 genes encoded hypothetical proteins. Further bioinformatic analysis demonstrated that 1350 genes were upregulated and 765 downregulated at least with half (four) of the utilizing EOs. A microarray investigation has confirmed the main impact of EOs to metabolic processes in P. rubens involved in vital functions. Presumably, this is the first time that a microarray hybridization analysis was performed in order to evaluate the gene expression of P. rubens exposed to various EOs.

A Study among the Genotype, Functional Alternations, and Phenotype of 9 SCN1A Mutations in Epilepsy Patients.

Mutations in the voltage-gated sodium channel Nav1.1 (SCN1A) are linked to various epileptic phenotypes with different severities, however, the consequences of newly identified SCN1A variants on patient phenotype is uncertain so far. The functional impact of nine SCN1A variants, including five novel variants identified in this study, was studied using whole-cell patch-clamp recordings measurement of mutant Nav1.1 channels expressed in HEK293T mammalian cells. E78X, W384X, E1587K, and R1596C channels failed to produce measurable sodium currents, indicating complete loss of channel function. E788K and M909K variants resulted in partial loss of function by exhibiting reduced current density, depolarizing shifts of the activation and hyperpolarizing shifts of the inactivation curves, and slower recovery from inactivation. Hyperpolarizing shifts of the activation and inactivation curves were observed in D249E channels along with slower recovery from inactivation. Slower recovery from inactivation was observed in E78D and T1934I with reduced current density in T1934I channels. Various functional effects were observed with the lack of sodium current being mainly associated with severe phenotypes and milder symptoms with less damaging channel alteration. In vitro functional analysis is thus fundamental for elucidation of the molecular mechanisms of epilepsy, to guide patients' treatment, and finally indicate misdiagnosis of SCN1A related epilepsies.

Structural and Functional Impact of Seven Missense Variants of Phenylalanine Hydroxylase.

The molecular genetics of well-characterized inherited diseases, such as phenylketonuria (PKU) and hyperphenylalaninemia (HPA) predominantly caused by mutations in the phenylalanine hydroxylase (PAH) gene, is often complicated by the identification of many novel variants, often with no obvious impact on the associated disorder. To date, more than 1100 PAH variants have been identified of which a substantial portion have unknown clinical significance. In this work, we study the functionality of seven yet uncharacterized PAH missense variants p.Asn167Tyr, p.Thr200Asn, p.Asp229Gly, p.Gly239Ala, p.Phe263Ser, p.Ala342Pro, and p.Ile406Met first identified in the Czech PKU/HPA patients. From all tested variants, three of them, namely p.Asn167Tyr, p.Thr200Asn, and p.Ile406Met, exerted residual enzymatic activity in vitro similar to wild type (WT) PAH, however, when expressed in HepG2 cells, their protein level reached a maximum of 72.1% ± 4.9%, 11.2% ± 4.2%, and 36.6% ± 7.3% compared to WT PAH, respectively. Remaining variants were null with no enzyme activity and decreased protein levels in HepG2 cells. The chaperone-like effect of applied BH4 precursor increased protein level significantly for p.Asn167Tyr, p.Asp229Gly, p.Ala342Pro, and p.Ile406Met. Taken together, our results of functional characterization in combination with in silico prediction suggest that while p.Asn167Tyr, p.Thr200Asn, and p.Ile406Met PAH variants have a mild impact on the protein, p.Asp229Gly, p.Gly239Ala, p.Phe263Ser, and p.Ala342Pro severely affect protein structure and function.

Homogentisate 1,2-dioxygenase (HGD) gene variants, their analysis and genotype-phenotype correlations in the largest cohort of patients with AKU.

Alkaptonuria (AKU) is a rare metabolic disorder caused by a deficient enzyme in the tyrosine degradation pathway, homogentisate 1,2-dioxygenase (HGD). In 172 AKU patients from 39 countries, we identified 28 novel variants of the HGD gene, which include three larger genomic deletions within this gene discovered via self-designed multiplex ligation-dependent probe amplification (MLPA) probes. In addition, using a reporter minigene assay, we provide evidence that three of eight tested variants potentially affecting splicing cause exon skipping or cryptic splice-site activation. Extensive bioinformatics analysis of novel missense variants, and of the entire HGD monomer, confirmed mCSM as an effective computational tool for evaluating possible enzyme inactivation mechanisms. For the first time for AKU, a genotype-phenotype correlation study was performed for the three most frequent HGD variants identified in the Suitability Of Nitisinone in Alkaptonuria 2 (SONIA2) study. We found a small but statistically significant difference in urinary homogentisic acid (HGA) excretion, corrected for dietary protein intake, between variants leading to 1% or >30% residual HGD activity. There was, interestingly, no difference in serum levels or absolute urinary excretion of HGA, or clinical symptoms, indicating that protein intake is more important than differences in HGD variants for the amounts of HGA that accumulate in the body of AKU patients.

Type 3 inositol 1,4,5-trisphosphate receptor has antiapoptotic and proliferative role in cancer cells.

Although the involvement of type 1 (IP3R1) and type 2 (IP3R2) inositol 1,4,5-trisphosphate receptors in apoptosis induction has been well documented in different cancer cells and tissues, the function of type 3 IP3R (IP3R3) is still elusive. Therefore, in this work we focused on the role of IP3R3 in tumor cells in vitro and in vivo. We determined increased expression of this receptor in clear cell renal cell carcinoma compared to matched unaffected part of the kidney from the same patient. Thus, we hypothesized about different functions of IP3R3 compared to IP3R1 and IP3R2 in tumor cells. Silencing of IP3R1 prevented apoptosis induction in colorectal cancer DLD1 cells, ovarian cancer A2780 cells, and clear cell renal cell carcinoma RCC4 cells, compared to apoptosis in cells treated with scrambled siRNA. As expected, silencing of IP3R3 and subsequent apoptosis induction resulted in increased levels of apoptosis in all these cells. Further, we prepared a DLD1/IP3R3_del cell line using CRISPR/Cas9 gene editing method. These cells were injected into nude mice and tumor's volume was compared with tumors induced by DLD1 cells. Lower volume of tumors originated from DLD1/IP3R3_del cells was observed after 12 days, compared to wild type DLD1 cells. Also, the migration of these cells was lesser compared to wild type DLD1 cells. Apoptosis under hypoxic conditions was more pronounced in DLD1/IP3R3_del cells than in DLD1 cells. These results clearly show that IP3R3 has proliferative and anti-apoptotic effect in tumor cells, on contrary to the pro-apoptotic effect of IP3R1.