| With the development of epigenetics,long non-coding RNA(lncRNA)is more and more studied in the development of tumorigenesis.Its expression level is related to the diagnosis,treatment and prognosis of tumor.The role of IncRNA in the molecular mechanism of environmental chemical carcinogenesis is unclear.The first discovery of IncRNA,MALAT1,is a carcinogenic lncRNA that is highly expressed in a variety of cancers,which regulates cell proliferation,apoptosis and migration,but is not reported in relation to arsenic carcinogenesis.Malignant tumors in the case of adequate oxygen can still produce high-efficiency glycolysis,for the survival of tumor cells to provide a certain material basis.Inflammation is a very important and important basic pathology,and studies have shown that most of the disease in the body originates from the onset of inflammatory responses.Long-term chronic inflammatory induces secretion of much inflammatory mediators by cascade amplification effect,which leading to micro-environmental disorders,and then induced cell proliferation,gene mutation,making the gene mutation of the cell repair dysfunction,and ultimately lead to cancer.At present,the relationship between the Warburg effect and the inflammatory response has aroused the concern of the majority scholars,and found that the final product of the Warburg effect of lactic acid induced inflammation and cell metastasis and invasion ability,which is thought that Warburg effect and inflammatory response are involved in the development of various malignant tumors,but the specific mechanism still needs further exploration.Although the Warburg effect and inflammatory response are reported less in body damage caused by exposure to environmental chemicals,we hypothesize that the Warburg effect and its inflammatory response may be involved in arsenic-induced malignant transformation of cells and their tumor progression.Therefore,in-depth study of arsenic exposure caused by Warburg effect and inflammatory response and its relationship with the malignant transformation of cells and specific mechanisms,which for the understanding of the body caused by arsenic damage and tumor molecular mechanisms to find early biological damage markers,it is great theoretical and practical value to find out that the prevention and control measures are of great significance.Part I Role and molecular mechanism of LncRNA MALAT1 in malignant transformation of L-02 cells induced by arseniteObjectiveTo investigate the effect of arsenite on metastasis-associated lung adenocarcinoma transcript 1(MALAT1),and to explore the role of IncRNA MALAT1 in arsenite-induced malignant transformation of cells.MethodsIn this study,we selected human normal liver(L-02)cells to study the establishment of arsenite-induced L-02 cell proliferation and malignant transformation model;soft agar colony formation and nude mice tumorigenesis test are used to detect cell malignancy.RT-PCR and qRT-PCR were used to detect the expression of IncRNA MALAT1.To investigate the role of IncRNA MALAT1 in arsenite-induced malignant transformation of cells by lentivirus stable transfection technique.Results1.Effects of arsenite on the malignant transformation of L-02 cellsL-02 cells were exposed to 0,1,2,5,10 or 20 pM arsenite for 24,48 or 72 h respectively.The results showed that 2 μM arsenite-induced L-02 cell proliferation increased most significantly.Then,the L-02 cells were treated with 2 μM arsenite for 30 generations.The results showed that 2 μM arsenite chronic treatment of L-02 cells to form clonal colonies and nude mice can form a tumor subcutaneous.Suggesting that 2 μM arsenite causes L-02 cell proliferation and malignant transformation.2.Effects of arsenite on IncRNA MALAT1 in L-02 cellsL-02 cells were exposed to 0 or 2 μM arsenite for 30 generations.The results showed that 2 μM arsenite increased IncRNA H19,HOTAIR and MALAT1 in L-02 cells,and the increase of lncRNA MALAT1 was the most obvious.The L-02 cells were further exposed to 0 or 2 μM arsenite for 0,3,6,12 or 24 h.The results showed that the level of lncRNA MALAT1 was gradually increased with the prolongation of acute treatment time.Suggesting that arsenite can induce L-02 cells IncRNA MALAT1 levels increased.3.Effect of LncRNA MALAT1 on malignant transformation of L-02 cells induced by arseniteArsenite-transformed L-02 cells were transfected with 10 μL control or sh-MALAT1 Virus,and found that inhibition of IncRNA MALAT1 levels can reduce the number of large colonies,the volume of the tumor in nude mice,and the ability of invasion and migration.Indicating that IncRNA MALAT1 plays an important role in the malignant transformation and tumorigenesis of L-02 induced by arsenite.ConclusionArsenite can induce L-02 cells proliferation and malignant transformation;and induce the high expression of lncRNA MALAT1;high expression of lncRNA MALAT1 plays an important role in in the malignant transformation of L-02 induced by arsenite.Part II Role and molecular mechanism of LncRNA MALAT1-induced Warburg effect in malignant transformation of L-02 cells induced by arseniteObjectiveTo investigate the role of lncRNA MALATI in arsenite-induced Warburg effect and its mechanism and the effect of Warburg effect on arsenite-induced malignant transformation of cells.MethodsIn this study,we successfully constructed the malignant transformation of L-02 cell model induced by arsenite.The lactate assay kit and glucose assay kit were used to determine the levels of lactate secretion and glucose consumption.The mitochondrial damage of L-02 cells was detected by transmission electron microscopy.Western blot and PCR were used to detect the expression of HIFs and hydroxylation of HIFs.The combination of VHL and HIF-α was detected by immunoprecipitation assay.By applying siRNA interference technology to investigate the role of lncRNA MALAT1 in the regulation of HIF-1α and the effect of arsenite on Warburg effect.And the role of the Warburg effect in the malignant transformation of L-02 cells was examined by colony formation assay and nude mice tumorigenesis assay.Results1.Effects of arsenite on Warburg effect of L-02 cellsL-02 cells were exposed to 0 or 2 μM arsenite for 0,3,6,12 or 24 h.The results showed that arsenite increases lactate secretion and glucose consumption,as well as the glycolytic related genes HK-2,Eno-1 and Glut-4 expression.L-02 cells were exposed to 0 or 2 μM arsenite for 0,10,20 or 30 generations.The results showed that arsenite increases lactate secretion,glucose consumption and the expression of glycolytic related genes,and found mitochondrial damage.Suggesting that arsenite can induce energy metabolism patterns changed in L-02 cells,and induce the Warburg effect.2.Effects of IncRNA MALAT1 on Warburg effect of L-02 cells induced by arseniteL-02 cells were treated with 100 ppm con-siRNA or IncRNA MALAT1-siRNA for 24 h,and the cells were exposed to 0 or 2μM arsenite for 24 h.Our results showed that knockdown of IncRNA MALAT1 attenuated arsenite-induced lactate production,glucose uptake and expression of glycolytic genes.These data indicate that IncRNA MALATI functions as a mediator of arsenite-enhanced glycolysis.3.Effects of IncRNA MALAT1 on the levels of HIFs induced by arsenite in L-02 cells.L-02 cells were exposed to 0 or 2 μM arsenite for 0,3,6,12 or 24 h,and the levels of HIF-la and HIF-2a were gradually increased.Further,L-02 cells were exposed to 0 or 2 μM arsenite for 0,10,20 or 30 generations,and the expression levels of HIF-la and HIF-2a were gradually increased.Suggesting that arsenite can induce the levels of HIF-la and HIF-2a in L-02 cells.L-02 cells were treated with 100 ppm con-siRNA or IncRNA MALAT1-siRNA for 24 h,and the cells were exposed to 0 or 2 μM arsenite for 24 h.The results showed that knockdown of IncRNA MALAT1 attenuated arsenite-increased HIF-la and HIF-2a protein levels,but has no effect on mRNA levels.Further,we found that inhibition of IncRNA MALAT1 reverse the block in degradation of ubiquitinated HIF-la and HIF-2a induced by 2 μM arsenite,thereby the levels of HIF-la and HIF-2a are reduced.These data suggest that LncRNA MALAT1 stabilizes its expression by inhibiting ubiquitination degradation of HIF-la and HIF-2a.4.The roles of IncRNA MALAT1 regulated HIF-la in the Warburg effect of L-02 induced by arseniteL-02 cells were treated with 10 nM con-siRNA,HIF-la-siRNA or HIF-2a-siRNA for 24 h,and then exposed to 0 or 2 pM arsenite for 24 h.The results showed that the inhibition of HIF-la blocked 2 μM arsenite-induced Warburg effect,however inhibition of HIF-2a did not occur this phenomenon.Further,L-02 cells were co-transfected with IncRNA MALAT1 siRNA and HIF-la plasmid or control plasmid for 24 h,then cells exposed to arsenite for 24 h.The results showed that ectopic expression of HIF-la reversed the effect of knockdown of IncRNA MALAT1 on the Warburg effect.Therefore,these data indicate that the mediating effect of IncRNA M ALAT1 on arsenite-enhanced glycolysis is dependent on HIF-la.5.The roles of the Warburg effect in malignant transformation of L-02 cells induced by arseniteL-02 cells were treated with 3 mM glycolytic inhibitor 2-DG for 3 h,and then cells were treated with 0 or 2 μM arsenite for 24 to 48 h(1 generation),which was continued for 30 generations.The results showed that inhibition of glycolysis level prevented 2 μM arsenite-induced soft agar colony formation,nude mice subcutaneous tumor formation and reduce cell metastasis and invasive ability.Indicating that the Warburg effect plays an important role in the malignant transformation and tumorigenesis of L-02 cells induced by arsenite.ConclusionArsenite can increase the levels of lactate production,glucose consumption,and increases glycolytic genes overexpression,and induce mitochondrial injury,which induce the Warburg effect.LncRNA MALAT1 stabilizes its expression by inhibiting ubiquitination degradation of HIF-a.LncRNA MALAT1 regulates HIF-la play an important role in arsenite-induced Warburg effect of L-02 cells.All these indicate that IncRNA MALAT1 regulating HIF-la-induced Warburg effect plays an important role in the malignant transformation of L-02 cells induced by arsenite.Part Ⅲ Effect and mechanism of Warburg effect regulating inflammatory reaction on malignant transformation of L-02 cells induced by arseniteObjectiveTo investigate the role of HIF-la-mediated Warburg effect in arsenite-induced inflammatory response and its mechanism and the role of inflammatory response in arsenite-induced malignant transformation of cells.MethodsIn this study,we successfully constructed the malignant transformation of L-02 cell model induced by arsenite.ELISA was used to determine the levels of IL-6,IL-8,and TNF-a in L-02 cells exposed.Western blot and PCR were performed to determine the levels of cytokines and MCT-4.The reporter assays were performed to determine if HIF-la directly regulate MCT-4 transcriptional activity.By using inhibitor,siRNA interference and human recombinant IL-6/8 to investigate the effect of HIF-la-mediated Warburg effect on arsenite-induced inflammatory response and inflammatory response-induced malignant transformation.Results1.Effects of arsenite on inflammation of L-02 cellsL-02 cells were treated by 0 or 2 μM arsenite for 3,6,12,or 24h,respectively;results showed that arsenite increases the mRNA and secretion levels of pro-inflammatory cytokines IL-6,IL-8 and TNF-a.L-02 cells were exposed to 0 or 2μM arsenite for 0,10,20 or 30 generations.Our data showed that the mRNA and secretion levels of IL-6,IL-8 and TNF-a were increased in arsenite-transformed L-02 cells.These results suggest that arsenite induced inflammation in L-02 cells.2.The roles of the Warburg effect in inflammation of L-02 cells induced by arseniteL-02 cells were treated with 3 mM glycolytic inhibitor 2-DG for 3 h,and then cells were treated with 0 or 2 μM arsenite for 24 to 48 h(1 generation),which was continued for 30 generations.The results showed that inhibition of glycolysis level blocked the 2 μM arsenite-induced IL-6,IL-8 and TNF-a increased.These results suggest that Warburg effect is involved in inflammation induced by arsenite.3.The roles of HIF-1a in the levels of MCT-4 in L-02 cells exposed to arseniteL-02 cells were exposed to 0 or 2 μM arsenite for 0,3,6,12 or 24 h;our data showed that arsenite increases the expression of MCT-4.L-02 cells were exposed to 0 or 2 μM arsenite for 0,10,20 or 30 generations.The expression level of MCT-4 in L-02 cells was gradually increased.L-02 cells were treated with 10 nM con-siRNA or HIF-la-siRNA for 24 h and then exposed to 0 or 2 μM arsenite for 24 h.The results showed that knockdown of HIF-la attenuated arsenite-increased MCT-4 protein levels.Further,the reporter assays found that HIF-1α can directly regulate MCT-4 expression.These results indicate that HIF-la promotes the high expression of MCT-4 in L-02 cells exposed to arsenite.4.The roles of MCT-4-mediated inflammation in malignant transformation of L-02 cells induced by arseniteL-02 cells were exposed to 0 or 2 μM arsenite for 30 generations,then cells were treated by 10 nM MCT-4-siRNA or con-siRNA for 24 h;results showed that silencing MCT-4 reduced the mRNA and secretion levels of pro-inflammatory cytokines IL-6 and IL-8,indicating that MCT-4 plays an important role in arsenite-induced inflammatory response of L-02 cells.Furthermore,10 ng/mL of human recombinant cytokines IL-6 and IL-8 were further cultured in 10 nM con-siRNA or MCT-4-siRNA-treated malignant transformed L-02 cells for 24 h;these data showed that silencing MCT-4 reduced anchorage independent growth and the ability of invasion and migration of arsenite-transformed L-02 cells,but the decrease of which was reversed by adding recombinant IL-6 and IL-8.All these data suggest that MCT-4 regulated IL-6 and IL-8 are involved in the arsenite-induced transformation of L-02 cells.5.The roles of Warburg effect-mediated inflammation in malignant transformation of L-02 cells induced by arseniteL-02 cells were treated with 3 mM glycolytic inhibitor 2-DG for 3 h,and then cells were treated with 0 or 2 μM arsenite for 24 to 48 h(1 generation),which was continued for 30 generations,and applied 10 ng/mL human recombinant IL-6 and IL-8 proteins for 24 h;these data showed that inhibition of glycolysis reduced anchorage independent growth and the ability of invasion and migration of arsenite-transformed L-02 cells,but the decrease of which was reversed by adding recombinant IL-6 and IL-8.All these data suggest that Warburg effect regulated IL-6 and IL-8 are involved in the arsenite-induced transformation of L-02 cells.6.Levels of lactic acid and inflammatory cytokines in arsenic poisoning groups and their correlation with hair arsenic.Collecting different levels of arsenic poisoning population serum,the levels of lncRNA MALAT1,lactate and inflammatory cytokine IL-6,IL-8 were detected.These results showed that with the increase of arsenic poisoning,the levels of lactate and IL-6,IL-8 gradually increased,and a statistically significant positive correlation was observed between serum lactate and cytokines IL-6,IL-8.Suggesting that elevated lactate and inflammatory cytokines in the body are associated with long-term exposure to arsenic.ConclusionArsenite induces induces the inflammatory reaction and the Warburg effect plays an important role in the inflammatory response of L-02 cells induced by arsenite;HIF-la directly regulates MCT-4 is involved in the inflammatory response of L-02 cells induced by arsenite.MCT-4 regulating inflammatory response plays an important role in the malignant transformation of L-02 cells induced by arsenite.Part IV LncRNA MALAT involved in the development of hepatocarcinogenesis of CD1 mice induced by arseniteObjectiveTo investigate the effect of arsenite on the occurrence of hepatocellular carcinoma,Warburg effect and inflammatory response in mice treated with "placental exposure + whole-life exposure" to different levels of arsenite,and to explore the role of lncRNAMALAT1 in arsenite-induced hepatocarcinogenesis in mice.MethodsIn this study,CD1 mice were selected to establish an animal model of mice with hepatocellular carcinoma induced by different levels arsenite by the exposure manner of "placental exposure + whole-life exposure".Statistical analysis of mice survival and body weight changes.Histological examination by Hematoxylin Eosin(H&E)stains were used to determine the liver cancer changes in CD1 mice;statistics of the incidence of liver cancer.The lactate assay kit was used to determine the levels of lactate secretion in mice serum.ELISA and qRT-PCR were used to determine the levels of pro-inflammatory cytokines.qRT-PCR were used to determine the levels of lncRNA MALAT1.Immunochemistry and western blot were performed to determine the levels of HIFs.The expression of HIFs and lncRNA MALAT 1 was analyzed by Pearson correlation analysis.To investigate the role of lncRNA MALAT1 in arsenite-induced hepatocarcinogenesis in mice.Results1.Effects of different levels of arsenite on the basic situation of male and female mice and the occurrence of liver tumors.CD1 mice were fed with feed and free drinking water containing 0,20,40 and 80 ppm arsenite for 104 weeks according to the "placental exposure + whole-life exposure" method to establish liver cancer animals model with different levels of arsenite.The results showed that the survival rate and body weight of mice in the experimental group were lower than those in the control group.HE staining showed that the liver lobules of the control group are intact and the hepatocytes were neat and the nuclei were clearly visible.The normal hepatic lobule structure of the male and female mice exposed to arsenite was destroyed,showing that the cancer cells were polygonal,abnormalities,the nucleus increased,cytoplasm decreased,visible multiple deformed mononuclear,multinucleated giant cells and mitotic phase,also seen a small number of venous tumor thrombus.Histological manifestations of hepatocellular carcinoma,cholangiocarcinoma rare.The incidence of HCC was found in all progeny mice,and the incidence of hepatocellular carcinoma was significantly increased in whole-life exposure to arsenite,and the highest incidence was found in the high dose 80 ppm group.These results suggest that exposure to different levels of arsenite by "placental exposure + whole-life exposure" can induce the occurrence of liver tumors in male and female mice.2.Effects of different levels of arsenite on the lactate and inflammation of male and female mice.CD1 mice were fed with feed and free drinking water containing 0,20,40 and 80 ppm arsenite for 104 weeks according to the "placental exposure + whole-life exposure" method to establish liver cancer animals model with different levels of arsenite.The results showed that the levels of serum lactate in the experimental group were increased,and the levels of inflammatory cytokines in liver tissue and serum were increased,which indicated that exposure to different levels of arsenite by"placental exposure + whole-life exposure" could induce the Warburg effect and inflammatory response of male and female mice.3.Effect of different levels of arsenite on the expression of lncRNA MALAT1 and HIFs in male and female miceCD1 mice were fed with feed and free drinking water containing 0,20,40 and 80 ppm arsenite for 104 weeks according to the "placental exposure + whole-life exposure" method to establish liver cancer animals model with different levels of arsenite.The results showed that the levels of IncRNA MALAT1 in serum and liver tissue were increased in the experimental group;and the levels of HIF-la and HIF-2a were increased in liver tissue.There was a linear correlation between the level of lncRNA MALAT1 and HIF-la and HIF-2a in liver tissue.Indicating that arsenite-induced liver cancer in mice,may be associated with IncRNA MALAT1 and HIFs increased.ConclusionThe exposure to different levels of arsenite by "placental exposure + whole-life exposure" can induce Warburg effect,inflammatory response and the occurrence of mice liver tumors.The expression of lncRNA MALAT1 and HIF-la and HIF-2a in the liver tissue of mice is induced by different levels of arsenite,and the levels of rise may be related to the occurrence of the tumor.In summary,the novel findings of this study are as follows:1.Arsenite can induce L-02 cell proliferation and malignant transformation,and IncRNA MALAT1 plays an important role in arsenite-induced L-02 cell malignant transformation process and metastasis.2.LncRNA MALAT1 is responsible for ubiquitin-mediated stabilization of HIF-la protein,and induces HIF-a overexpression;then HIF-1α induces the Warburg effect,which leads to the increase of lactate level,and eventually promotes L-02 cells malignant transformation.3.HIF-la directly regulates MCT-4 expression involved in arsenite-induced L-02 cells lactate secretion increased,which induces the formation of inflammation,and promotes the malignant transformation and metastasis.4.The exposure to arsenite by "placental exposure + whole-life exposure" can induce the Warburg effect,inflammatory response and liver tumorigenesis in mice,which may be related to the increase of lncRNA MALAT1 and HIFs. |
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