Cx43 Gene Research Articles

Mutations of the Cx43 Gene in Non-Small Cell Lung Cancer: Association with Aberrant Localization of Cx43 Protein Expression and Tumor Progression.

Background and Objectives: The Connexin43 (Cx43) gene is a suspected tumor suppressor gene, as re-expressed wild-type Cx43 genes reduce the malignancy potential of tumor cells. However, the role of Cx43 gene expression in human lung tumorigenesis remains unclear. Materials and Methods: Tumor tissues from 165 primary lung cancer patients were collected to study Cx43 protein expression and gene mutations using immunohistochemistry and direct DNA sequencing. In addition, Cx43 genes with or without mutations were transfected to CL-3 human lung cancer cells to confirm the function of these mutant forms of the Cx43 gene. Results: Aberrant localization of Cx43 protein in the nucleus and cytoplasm of tumor cells was detected in 14 out of 165 non-small cell lung cancer (NSCLC) patients. Mutations in the Cx43 gene were also found in patients with aberrant Cx43 localization, and transfection of these mutant genes into lung cancer cells enhanced their proliferation. Conclusions: To our knowledge, this is the first study to demonstrate Cx43 gene mutations in human lung neoplasm, supporting the hypothesis that Cx43 may function as a tumor suppressor in some lung cancer patients. Additionally, the findings suggest an association between aberrant localization of Cx43 protein expression and tumor progression.

Tamoxifen-induced alterations in the expression of connexin 43 in the chicken ovary

Connexin 43 (Cx43) is a gap junction protein that participates in small molecule exchange between adjacent cells. It is a predominant Cx within the mammalian ovary, where is associated with proper follicle development. The expression and regulation of Cx43 in the chicken ovary is largely unknown. The aim of the present study was to examine the expression of the Cx43 gene (GJA1) and protein as well as the immunolocalization of Cx43 in the laying hen ovary in relation to follicle development, and to examine how tamoxifen (TMX; an estrogen receptor modulator) treatment affects these factors. qRT-PCR and western blotting demonstrated differences in Cx43 mRNA transcript and protein abundances in ovarian white follicles, yellowish follicles, small yellow follicles, and the largest yellow preovulatory follicles (F3–F1). In general, Cx43 was more abundant in hierarchical than prehierarchical follicles and in granulosa cells compared with theca cells. Further, the response to TMX treatment depended on the stage of follicle development and the layer of the follicular wall. Ovarian regression following TMX treatment was accompanied by an increase in Cx43 expression in most ovarian tissues, which may impact the formation and function of Cx43 hemichannels. Overall, our results showed, for the first time, the differences in Cx43 mRNA and protein levels between ovarian follicles, suggesting the potential involvement of this gap junction protein in the regulation of ovarian follicle development and function. In addition, the results indicate a possible role for estradiol in regulation of Cx43 transcription and/or translation in the chicken ovary. Understanding the contribution of Cx43 in mechanisms underlying ovarian follicle development may be of considerable importance for poultry egg production.

Protector Role of Cx30.2 in Pancreatic β-Cell against Glucotoxicity-Induced Apoptosis.

RIN-m5F cells were cultured in 5 mM D-glucose (normal) or 30 mM D-glucose (high glucose) for 24 h. Cx30.2 siRNAs was used to downregulate Cx30.2 expression. Apoptosis was measured by means of TUNEL, an annexin V staining method, and the cleaved form of the caspase-3 protein was determined using Western blot. High glucose did not induce apoptosis in RIN-m5F β cells after 24 h; interestingly, high glucose increased the Cx30.2 total protein levels. Moreover, this work found that the downregulation of Cx30.2 expression in high glucose promoted apoptosis in RIN-m5F cells. The data suggest that the upregulation of Cx30.2 protects β cells from hyperglycemia-induced apoptosis. Furthermore, Cx30.2 may be a promising avenue of therapeutic investigation for the treatment of glucose metabolic disorders.

TBX3 transfection and nodal signal pathway inhibition promote differentiation of adipose mesenchymal stem cell to cardiac pacemaker-like cells.

Mesenchymal stem cells (MSCs) are known as one of the best candidate cells to produce cardiac pacemaker-like cells (CPLCs). Upregulation of TBX3 transcription factor and inhibition of the nodal signal pathway have a significant role in the formation of cardiac pacemaker cells such as sinoatrial and atrioventricular nodes, which initiate the heartbeat and control the rhythm of heart contractions. This study aimed to confirm the effects of transfection of TBX3 transcription factor and inhibition of the nodal signal pathway on differentiating adipose-derived MSCs (AD-MSCs) to CPLCs. AD-MSCs were characterized using flow cytometry and three-lineage differentiation staining. The transfection of TBX3 plasmid was carried out using lipofectamine, and inhibition of the nodal signal pathway was done using the small-molecule SB431542. The morphology of the cells was observed using a light microscope. Pacemaker-specific markers, including TBX3, Cx30, HCN4, HCN1, HCN3, and KCNN4, were evaluated using the qRT-PCR method. For protein level, TBX3 and Cx30 were evaluated using ELISA and immunofluorescence staining. The electrophysiology of cells was evaluated using a patch clamp. The TBX3 expression in the TBX3, SM, and TBX + SM groups significantly higher (p < 0.05) compared to the control group and cardiomyocytes. The expression of Cx40 and Cx43 genes were lower in TBX3, SM, TBX + SM groups. In contrast, Cx30 gene showed higher expression in TBX3 group. The expression HCN1, HCN3, and HCN4 genes are higher in TBX3 group. The transfection of TBX3 and inhibition of the nodal signal pathway by small-molecule SB431542 enhanced differentiation of AD-MSCs to CPLCs.

Connexin43 in mesenchymal lineage cells regulates body adiposity and energy metabolism in mice.

Connexin43 (Cx43) is the most abundant gap junction protein present in the mesenchymal lineage. In mature adipocytes, Cx43 mediates white adipose tissue (WAT) beiging in response to cold exposure and maintains the mitochondrial integrity of brown adipose tissue (BAT). We found that genetic deletion of Gja1 (Cx43 gene) in cells that give rise to chondro-osteogenic and adipogenic precursors driven by the Dermo1/Twist2 promoter led to lower body adiposity and partial protection against the weight gain and metabolic syndrome induced by a high-fat diet (HFD) in both sexes. These protective effects were related to increased locomotion, fuel utilization, energy expenditure, nonshivering thermogenesis, and better glucose tolerance in conditionally Gja1-ablated mice. Accordingly, Gja1-mutant mice exhibited reduced adipocyte hypertrophy, partially preserved insulin sensitivity, increased BAT lipolysis, and decreased whitening under HFD. This metabolic phenotype was not reproduced with more restricted Gja1 ablation in differentiated adipocytes, suggesting that Cx43 in adipocyte progenitors or other targeted cells restrains energy expenditures and promotes fat accumulation. These results reveal what we believe is a hitherto unknown action of Cx43 in adiposity, and offer a promising new pharmacologic target for improving metabolic balance in diabetes and obesity.

Loss of Function of the Retinoblastoma Gene Affects Gap Junctional Intercellular Communication and Cell Fate in Osteoblasts.

Loss of function of the Retinoblastoma gene (RB1) due to mutations is commonly seen in human osteosarcomas. One of the Rb1 gene functions is to facilitate cell fate from mesenchymal stem cells to osteoblasts and prevent adipocyte differentiations. In this study, we demonstrate that a stable reduction of Rb1 expression (RbKD) in murine osteoblasts causes them to express higher levels of PPAR-ɣ and other adipocyte-specific transcription factors while retaining high expression of osteoblast-specific transcription factors, Runx2/Cbfa1 and SP7/Osterix. Inhibition of gap junctional intercellular communication (GJIC) in osteoblasts is another mechanism that causes osteoblasts to transdifferentiate to adipocytes. We found that preosteoblasts exposed to osteoblast differentiating media (DP media) increased GJIC. RbKD cells showed reduced GJIC along with a reduction in expression of Cx43, the protein that mediates GJIC. Other membrane associated adhesion protein Cadherin 11 (Cad11) was also decreased. Since PPAR-ɣ is increased with Rb1 loss, we wondered if the reduction of this transcription factor would reverse the changes observed. Reduction of PPAR-ɣ in control osteoblasts slightly increased bone-specific expression and reduced adipocytic expression as expected along with an increase in Cad11 and Cx43 expression. GJIC remained high and was unaffected by a reduction in PPAR-ɣ in control cells. Knockdown of PPAR-ɣ in RbKD cells reduced adipocyte gene expression, while osteoblast-specific expression showed improvement. Cx43, Cad11 and GJIC remained unaffected by PPAR-ɣ reduction. Our observations suggest that increased PPAR-ɣ that happens with Rb1 loss only affects osteoblast-adipocyte-specific gene expression but does not completely reverse Cx43 gene expression or GJIC. Therefore, these effects may represent independent events triggered by Rb1loss and/or the differentiation process.

Connexin43 overexpression promoted ferroptosis and increased myocardial vulnerability to ischemia-reperfusion injury in type 1 diabetic mice.

Enhancement of Connexin43 (Cx43) and ferroptosis are respectively associated with the exacerbation of myocardial ischemia-reperfusion injury (MIRI) in diabetes. Myocardial vulnerability to ischemic insult has been shown to vary during early and later phases of diabetes in experimental settings. Whether or not Connexin43 (Cx43) and ferroptosis interplay during MIRI in diabetes is unknown. We, thus, aimed to investigate whether or not the content of myocardial Cx43 may be attributable to myocardial vulnerability to MIRI at different stages of diabetes and also to explore the potential interplay between Cx43 and ferroptosis in this pathology. Age-matched control and subgroups of Streptozotocin-induced diabetic mice were subjected to MIRI induced by 30 minutes coronary artery occlusion and 2 hours reperfusion respectively at 1, 2 and 5 weeks of diabetes. Rat cardiac H9C2 cells were exposed to high glucose (HG) for 48h in the absence or presence of Cx43 gene knockdown followed by hypoxia/reoxygenation (HR) respectively for 6 and 12 hours. Post-ischemic myocardial infarct size was reduced in 1 and 2 weeks DM mice concomitant with enhanced GPX4 and reduced cardiac Cx43 and ferroptosis as compared to control. By contrast, cardiac GPX4 was significantly reduced while Cx43 increased at DM 5 weeks (D5w) which was correspondent to significant increases in ferroptosis and myocardial infarction. Post-ischemic cardiac function was improved in 1 and 2 weeks but worsened in 5w DM mice as compared with non-diabetic control. GAP19 (Cx43 inhibitor) significantly attenuated ferroptosis and reduced myocardial infarction in D5w mice. Erastin (ferroptosis activator) reversed the cardioprotective effect of GAP19. In vitro, HR significantly reduced cell viability accompanied with reduced GPX4 but elevated Cx43 expression, MDA production and ferroptosis. Cx43 gene knockdown in H9C2 resulted in a significant increase in GPX4, reduction in MDA and ferroptosis, and subsequently reduced post-hypoxic cell viability. The beneficial effects of Cx43 gene knock-down was minified or eliminated by Erastin. It is concluded that Cx43 overexpression exacerbates MIRI under diabetic conditions via promoting ferroptosis, while its down-regulation at early state of diabetes is attributable to enhanced myocardial tolerance to MIRI.

Optimization of the cotransfection of SERCA2a and Cx43 genes for myocardial infarction complications

As our previous study showed, the therapeutic effect of two genes (SERCA2a and Cx43) on heart failure after myocardial infarction (MI) was greater than that of single gene (SERCA2a or Cx43) therapy for bone marrow stem cell (BMSC) transplantation. Based on previous research, the aim of this study was to investigate the optimal ratio of codelivery of SERCA2a and Cx43 genes for MI therapy after biotinylated microbubble (BMB) transplantation via ultrasonic-targeted microbubble destruction (UTMD). Forty rats underwent left anterior descending (LAD) ligation and BMSC injection into the infarct and border zones. Four weeks later, the genes SERCA2a and Cx43 were codelivered at different ratios (1:1, 1:2 and 2:1) into the infarcted heart via UTMD. Cardiac mechanoelectrical function was determined at 4 wks after gene delivery, and the hearts of the rats were harvested for measurement of MI size and detection of SERCA2a and Cx43 expression. Q-PCR analysis of the expression of Nkx2.5 and GATA4 in the myocardial infarct zone and measurement of neovascularization in infarcted hearts. After comparing the therapeutic effects of different cogene ratios, the SERCA2a/Cx43–1:2 group showed remarkable cardiac electrical stability and strengthened the role of anti-arrhythmia. In conclusion, the optimum ratio of the SERCA2a/Cx43 gene is 1:2, which is advantageous for maintaining cardiac electrophysiological stability.

Cx43 regulates mechanotransduction mechanisms in human preterm amniotic membrane defects.

The effects of mechanical stimulation in preterm amniotic membrane (AM) defects were explored. Preterm AM was collected from women undergoing planned preterm caesarean section (CS) due to fetal growth restriction or emergency CS after spontaneous preterm prelabour rupture of the membranes (sPPROM). AM explants near the cervix or placenta were subjected to trauma and/or mechanical stimulation with the Cx43 antisense. Markers for nuclear morphology (DAPI), myofibroblasts (αSMA), migration (Cx43), inflammation (PGE2 ) and repair (collagen, elastin and transforming growth factor β [TGFβ1 ]) were examined by confocal microscopy, second harmonic generation, qPCR and biochemical assays. In preterm AM defects, myofibroblast nuclei were highly deformed and contractile and expressed αSMA and Cx43. Mechanical stimulation increased collagen fibre polarisation and the effects on matrix markers were dependent on tissue region, disease state, gestational age and the number of fetuses. PGE2 levels were broadly similar but reduced after co-treatment with Cx43 antisense in late sPPROM AM defects. TGFβ1 and Cx43 gene expression were significantly increased after trauma and mechanical stimulation but this response dependent on gestational age. Mechanical stimulation affects Cx43 signalling and cell/collagen mechanics in preterm AM defects. Establishing how Cx43 regulates mechanosignalling could be an approach to repair tissue integrity after trauma.

Mechanism Exploration of Environmental Pollutants on Premature Ovarian Insufficiency: a Systematic Review and Meta-analysis.

As a public health problem, premature ovarian insufficiency leads to infertility or sub-fertility. In addition to premature ovarian insufficiency (POI) increases the lifetime risk of bone fragility, cardiovascular disease, and cognitive impairment. To investigate the effects of environmental pollutants on the occurrence of POI and explore its mechanism, we conducted a computer search for articles published in electronic databases by December 13, 2022. Three reviewers independently examined all included studies and scored the qualities of included studies using the Newcastle-Ottawa Scale criteria. In this meta-analysis, eight clinical studies as well as ten preclinical findings showed a pooled OR of 2.331 and 95% CI of 1.968-2.760. This confirms that environmental pollutants, including POPs, heavy metals, PAEs, PAHs, cosmetic and pharmaceutical products, and cigarette smoke, are indeed significant risk factors for POI. In addition, it is demonstrated from the results of this study that signaling pathway of calcium and PI3K Akt and Xpnpep2, Col1, Col3, Col4, Cx43, Egr3, Tff1, and Ptgs2 genes may all be involved in the process. Environmental pollutants, including POPs, heavy metals, PAEs, PAHs, cosmetic and pharmaceutical products, and cigarette smoke, are indeed significant risk factors for POI.

Gene replacement therapy in two Golgi-retained CMT1X mutants before and after the onset of demyelinating neuropathy

X-linked Charcot-Marie-Tooth disease type 1 (CMT1X) is a demyelinating neuropathy resulting from loss-of-function mutations affecting the GJB1/connexin 32 (Cx32) gene. We previously showed functional and morphological improvement in Gjb1-null mice following AAV9-mediated delivery of human Cx32 driven by the myelin protein zero (Mpz) promoter in Schwann cells. However, CMT1X mutants may interfere with virally delivered wild-type (WT) Cx32. To confirm the efficacy of this vector also in the presence of CMT1X mutants, we delivered AAV9-Mpz-GJB1 by lumbar intrathecal injection in R75W/Gjb1-null and N175D/Gjb1-null transgenic lines expressing Golgi-retained mutations, before and after the onset of the neuropathy. Widespread expression of virally delivered Cx32 was demonstrated in both genotypes. Re-establishment of WT Cx32 function resulted in improved muscle strength and increased sciatic nerve motor conduction velocities in all treated groups from both mutant lines when treated before as well as after the onset of the neuropathy. Furthermore, morphological analysis showed improvement of myelination and reduction of inflammation in lumbar motor roots and peripheral nerves. In conclusion, this study provides proof of principle for a clinically translatable gene therapy approach to treat CMT1X before and after the onset of the neuropathy, even in the presence of endogenously expressed Golgi-retained Cx32 mutants.

The optimized method for mesenchymal stem cell differentiation into pacemaker-like cell

Link of Video Abstract: https://www.youtube.com/watch?v=HIbeTAOCFcABackground: Mesenchymal stem cells (MSCs) differentiate into pacemaker-like cells by activating TBX3 and downregulating Pitx2c by inhibiting the nodal signaling pathway. This optimization study aimed to determine the optimal conditions for differentiating MSCs into pacemaker-like cells. Methods: The differentiation of MSCs into pacemaker-like cells was carried out in three ways: transfecting TBX3 pcDNA into MSCs, inhibiting the nodal signaling pathway using the small molecule SB431542, and a combination of both methods. This optimization study was conducted in two batches. The first batch was an experiment with transfection carried out on the 5th day of differentiation and addition of 20 μM/mL of the small molecule SB431542. The second batch was an experiment with transfection carried out on the 3rd day of differentiation and the addition of 2 μM/mL of the small molecule SB431542. From the two batches, the expression patterns of TBX3, Cx30.2, Cx40, Cx43, HCN4, HCN1, HCN3, and KCNN4 genes were analyzed using the qRT-PCR method. Results: The results showed that the second batch had a better gene expression pattern in pacemaker-like cells than the first batch. These results were indicated by the increased expression of the TBX3, Cx30.2, HCN4, HCN1, HCN3, and KCNN4 genes. Furthermore, there was a decrease in Cx40 and Cx43 expression levels. Conclusion: The results showed that TBX3 transfection on the third day and a smaller dose (2 μM/mL) of small molecules and combination treatment induced differentiation from the initial MSC, leading to the expression of pacemaker-like cells.

Multi-omics analysis of expression profile and prognostic values of connexin family in LUAD.

Our study first explored the expression differences and prognostic significance of Cx genes in pan-cancer and then focused on LUAD. Our objectives were to conducted a comprehensive analysis of the expression profile, prognostic significance, genetic alterations, potential biological functions and drug sensitivity of the Connexin gene family in LUAD. We developed a comprehensive prognostic model for LUAD by combining risk scores with clinical features and created a nomogram to predict 1-, 3-, and 5-year overall survival. Using single-cell sequencing, we examined the expression and biological functions of the identified prognostic markers. Our risk model revealed that GJB2-5 play a critical role in the prognosis of LUAD patients, associated with many biological processes such as cell cycle, DNA damage, EMT, hypoxia, invasion, and metastasis. Furthermore, the connexin gene family is linked to transcriptional mechanisms such as the extracellular matrix (ECM), migration, mobility, angiogenesis, and the epithelial-mesenchymal transition (EMT) genetic program. The risk model can be used as a potential prognostic factor for LUAD patients and may provide new insights into cancer treatment from perspective of the expression of Cx genes.

The GJB2 (Cx26) Gene Variants in Patients with Hearing Impairment in the Baikal Lake Region (Russia).

The GJB2 (Cx26) gene pathogenic variants are associated with autosomal recessive deafness type 1A (DFNB1A, OMIM #220290). Direct sequencing of the GJB2 gene among 165 hearing-impaired individuals living in the Baikal Lake region of Russia identified 14 allelic variants: pathogenic/likely pathogenic-nine variants, benign-three variants, unclassified-one variant, and one novel variant. The contribution of the GJB2 gene variants to the etiology of hearing impairment (HI) in the total sample of patients was 15.8% (26 out of 165) and significantly differed in patients of different ethnicity (5.1% in Buryat patients and 28.9% in Russian patients). In patients with DFNB1A (n = 26), HIs were congenital/early onset (92.3%), symmetric (88.5%), sensorineural (100.0%), and variable in severity (moderate-11.6%, severe-26.9% or profound-61.5%). The reconstruction of the SNP haplotypes with three frequent GJB2 pathogenic variants (c.-23+1G>A, c.35delG or c.235delC), in comparison with previously published data, supports a major role of the founder effect in the expansion of the c.-23+1G>A and c.35delG variants around the world. Comparative analysis of the haplotypes with c.235delC revealed one major haplotype G A C T (97.5%) in Eastern Asians (Chinese, Japanese and Korean patients) and two haplotypes, G A C T (71.4%) and G A C C (28.6%), in Northern Asians (Altaians, Buryats and Mongols). The variable structure of the c.235delC-haplotypes in Northern Asians requires more studies to expand our knowledge about the origin of this pathogenic variant.

Effect of progesterone on in vitro meiotic maturation of canine oocytes associated with Cx37 and Cx43 gene expression

Progesterone (P4) concentrations in canines are exceptionally high in the periovulatory period. However, the mechanisms by which P4 modulates final oocyte development in dogs remain to be characterized. The aim of this study was to evaluate the effect of P4 on meiotic development related to the gene expression of connexin 37 (Cx37) and connexin 43 (Cx43) in the canine cumlus oocyte complexes (COCs). COCs were isolated from 120 canine ovaries after a routine ovariohysterectomy. In each experiment, groups of COCs retrieved from the antral follicles were subjected to in vitro maturation (IVM) for 72 h without (control) or with P4 (50 μg/mL and 100 μg/mL) or the P4 receptor antagonist, aglepristone (RU534 at 1 μM and 10 μM). Some of the COCs recovered (from each group) after 72 h of IVM were subjected to meiotic evaluation; the remaining COCs, and those not subjected to IVM, were used to analyze the gene expression of Cx37 and Cx43 by qPCR. The results were evaluated using ANOVA. The addition of P4 increased (P < 0.05) the meiotic development compared to that in the control or aglepristone groups. The highest (P < 0.05) percentage of oocytes in the MII stage was observed upon P4 supplementation. In contrast, the highest percentage (P < 0.05) of oocytes arrested in the GV stage and the lowest (P < 0.05) percentages in the MII stage were observed for COCs cultured with aglepristone. Although a significant decrease in the mRNA levels of both connexins was observed after culturing, no effect on Cx37 and Cx43 gene expression was observed when exogenous P4 was added compared to those of the control group. However, COCs cultured with aglepristone exhibited higher (P < 0.05) expression of Cx37 and Cx43 than COCs in the control IVM-group, regardless of the concentration. In conclusion, our results suggest that a high dosage of P4 during IVM enhances the nuclear maturation of canine oocytes without altering the gene expression levels of Cx37 and Cx43. However, the increase in their expression upon treatment with a P4 antagonist indicates an in vivo role for this hormone in the endogenous modulation of both Cx37 and Cx43.

Low dose TGF-β1 can improve vohwinkel syndrome by promoting the proliferation of keratinocytes

Vohwinkel syndrome (VS) is a very rare autosomal dominant disorder that can cause disability and deformity in severe cases. Mutations of the LOR (loricrin) and GJB2 (Cx26) genes have been found in VS so far. Many studies have indicated that the differentiation and growth of epidermal keratinocytes are regulated by mutant Cx26, and it may explain the pathogenesis of VS. It has been found that transforming growth factor β1 (TGF-β1) expression was lower in G130V (OE1) and D66H (OE2) mutant keratinocytes in the VS model with GJB2 mutation as compared to normal keratinocytes (NC). TGF-β is a cytokine involved in the regulation of processes like cell proliferation and differentiation in different types of cells. At present, many in vitro studies focus on TGF- β 1 inhibition of keratinocyte growth.However, the relationship between TGF-β1 and VS remains unknown. This study aimed at elucidating the role and potential pathogenic mechanism of TGF-β in VS. The results indicated that the down-regulation expression of TGF-β1 in VS was linked to cell proliferation inhibition through p-Smad3/c-myc. In contrast, low-dose TGF-β1 treatment of VS keratinocytes can improve their proliferation inhibition and up-regulate the expression Cyclin D1. This suggests that low doses of TGF-β1 can improve the proliferation of VS and provide new insights into its treatment.

Effect of duloxetine as antidepressant on TM4 Sertoli cells: Evaluation of Bax and Cx43 gene expression

هدف: از چالش­های عمده بشریت افزایش افسردگی و اختلال عملکرد جنسی ناشی از داروهای ضدافسردگی است. با توجه به نقش سلول­های سرتولی در اسپرماتوژنز، پژوهش حاضر، اثر داروی دولوکستین را بر زنده­مانی، آپوپتوزیس و بیان ژن­های Bax و­ (Connexin 43) Cx43 در سلول­های سرتولی بررسی کرده است. مواد و روش‌‌ها: سلول­های TM4 در محیط DMEM/F12 حاوی %5/2 FBS، 5% سرم اسب و %1 پنی سیلین-استرپتومایسین کشت شدند. دولوکستین با دوزهای 30،60، 15، 5/7، 75/3 میکرو­گرم/ میلی­لیتر در زمان­های 24 تا 72 ساعت روی سلول­ها اثر داده شد. آزمون MTT جهت ارزیابی زنده­مانی سلول­ها، فلوسایتومتری جهت ارزیابی آپوپتوزیس و RT-qPCR جهت بررسی بیان ژن Bax (عامل پیش­برندۀ آپوپتوزیس) و Cx43 (ضروری برای اسپرماتوژنز) انجام شد. نتایج: دولوکستین در یک الگوی وابسته به دوز و زمان، بقای سلولی را کاهش داد. بر اساس داده­های MTT دوز IC50 15 میکروگرم/ میلی­لیتر در 48 ساعت محاسبه گردید (p≤0.05). آپوپتوزیس سلول­های TM4 نسبت به گروه کنترل در دوز میانه مهاری 15 میکروگرم/ میلی­لیتر دولوکستین، افزایش یافت (p≤0.01). RT-qPCR حاکی از افزایش بیان ژن­های Cx43 (p≤0.05) و Bax (p≤0.01) تحت تاثیر دولوکستین بود. نتیجه­گیری: با توجه به داده­های فلوسایتومتری و افزایش بیان ژن Bax، دولوکستین با القای آپوپتوزیس در سلول­های سرتولی می­تواند عاملی منفی و مخرب در پیشبرد اسپرماتوژنز در نظر گرفته شود. از سوی دیگر، دولوکستین با افزایش سطح بیان ژن Cx43، نقل و انتقالات مولکولی توسط اتصالات شکافدار را بین سلول­های سرتولی افزایش داده و می‫تواند باعث انتقال سیگنال­های پیش برنده آپوپتوزیس به سلول­های دودومان اسپرماتوژنیک و در نتیجه کاهش کیفیت اسپرماتوژنز ­شود.

Probing the interaction between metastatic breast cancer cells and osteoblasts in a thread-based breast-bone co-culture device.

Breast cancer metastasis to bone is a leading killer in breast cancer patients. A type I collagen-modified thread (Col-I@thread) was prepared for 3-dimensional cell culture and breast cancer bone metastasis co-culture device assembly. First, the coating of Col-I on nylon threads for promoting cell adhesion and growth was studied. Through SEM, XPS, and protein concentration measurements, it was found that the lyophilization method remarkably preserved the Col-I activity and the internal structure of the thread, thereby promoting cell attachment and proliferation. RNA-sequencing (RNA-Seq) and quantitative PCR analysis showed that osteoblast cells (MC3T3-E1) grown on Col-I@thread had elevated RUNX2, ALP, OPN, and Col-I gene expression to promote osteoblast differentiation. Single-cell analysis found that osteoblast MC3T3-E1 cells growing on Col-I@thread had higher Ca2+ secretion activity and mineralized nodules, suggesting robust cell activity and bone matrix formation than cells growing on 2D culture plates. Col-I@threads were knotted in an interdigital cross-finger frame to assemble the breast cancer-bone co-culture model. Confocal microscopy and flow cytometry tests quantified the invasive breast cancer cells. Moreover, the thread-based co-culture devices allowed us to isolate the invasive and non-invasive breast cancer cells to compare their molecular characteristics. qPCR results showed that expression of CX43, CXCR5, and CSPG4 genes was significantly increased in breast cancer cells with bone metastasis. Meanwhile, the expression of RUNX2 and OPG genes in osteoblasts was inhibited. The co-culture model based on the Col-I@thread mimics the bone tissue microenvironment to reveal the cross-talk between cancer cells and bone tissue. Moreover, the thread-based co-culture device is easy to fabricate and operate, providing a platform for exploring the cellular and molecular mechanisms of breast cancer bone metastasis, and holds potential for high-throughput screening of anti-breast cancer bone metastasis drugs.

Assessing E-Cadherin and Connexin 43 Gene Expressions in Colorectal Cancer

Objective: Dysregulation of cellular adhesion is one of the main mechanisms responsible for tumor initiation, proliferation, and survival. E-cadherin is a cell adhesion molecule associated with tissue invasion and metastasis in most epithelial cancers. Gap junctions are known as small molecular channels that allow communication between neighboring cells and consist of connexin molecules. Connexin 43 (Cx43) is a gap junction protein that plays a central role in cell-cycle regulation and has an important function in carcinogenesis. The present study aimed to evaluate the expression levels of E-cadherin and Cx43 in colorectal cancer patients using clinical and prognostic parameters. Materials and Methods: The quantitative real-time polymerase chain reaction (qRT-PCR) method was utilized to characterize the expression patterns of the E-cadherin and Cx43 genes in tumor and adjacent non-tumoral colon tissues from 32 colorectal cancer patients. Analysis of gene expression data was carried out using the delta-CT method. Results: The results show the expression level of Cx43 to decrease 14-fold in tumor tissue compared to normal tissue (p&lt;0.05). However, the study could find no significant difference with regard to E-cadherin expression. Conclusion: The research provides valuable clues to the elucidation of tumor development and metastatic processes for further studies.

Connexin 30 Deficiency Ameliorates Disease Progression at the Early Phase in a Mouse Model of Amyotrophic Lateral Sclerosis by Suppressing Glial Inflammation.

Connexin 30 (Cx30), which forms gap junctions between astrocytes, regulates cell adhesion and migration, and modulates glutamate transport. Cx30 is upregulated on activated astroglia in central nervous system inflammatory lesions, including spinal cord lesions in mutant superoxide dismutase 1 (mSOD1) transgenic amyotrophic lateral sclerosis (ALS) model mice. Here, we investigated the role of Cx30 in mSOD1 mice. Cx30 was highly expressed in the pre-onset stage in mSOD1 mice. mSOD1 mice with knockout (KO) of the Cx30 gene (Cx30KO-mSOD1 mice) showed delayed disease onset and tended to have an extended survival period (log-rank, p = 0.09). At the progressive and end stages of the disease, anterior horn cells were significantly preserved in Cx30KO-mSOD1 mice. In lesions of these mice, glial fibrillary acidic protein/C3-positive inflammatory astroglia were decreased. Additionally, the activation of astrocytes in Cx30KO-mSOD1 mice was reduced compared with mSOD1 mice by gene expression microarray. Furthermore, expression of connexin 43 at the pre-onset stage was downregulated in Cx30KO-mSOD1 mice. These findings suggest that reduced expression of astroglial Cx30 at the early disease stage in ALS model mice protects neurons by attenuating astroglial inflammation.