| Heavy metal cadmium is considered to be a non-essential element with serious toxicity to organisms.its accumulation in the body will cause harmful damage to bones,urinary system,reproductive system,cardiovascular system and immune system,and affect the survival and development of organisms.In the aquatic environment,once the stress of pollutants occurs,the endocrine activity of fish will be affected,resulting in metabolic disorders and immune system disorders,and then affect the normal physiological activities of fish.the main manifestations are as follows: slow growth rate,low immunity,decreased fecundity and so on,which seriously lead to death.Micro RNA(mi RNA)is a non-coding single-stranded small molecule RNA that has the function of post-transcriptional regulation of gene m RNA expression.Mi RNA involves many biological processes and plays an important role in cell proliferation,differentiation,development and autophagy.Autophagy is a physiological process that cells use to prevent the accumulation of toxic substances.It degrades misfolded proteins and damaged organelles by fusing autophagosomes with lysosomes.The results of full transcriptome showed that mi R-451 played an important role in the damage of carp head and kidney lymphocytes induced by cadmium poisoning enriching autophagy-related pathways.Target Scan website predicts that the target of mi R-451 is cacna1 ab.Mi R-451 is involved in a variety of physiological and pathological processes,but the specific mechanism of mi R-451 in cadmium-induced autophagy of carp head and kidney lymphocytes is still unknown.q RT-PCR(real-time fluorescence quantitative PCR),Western Blot(protein immunoblotting)and double luciferase reporter gene system were used to verify whether cacna1 ab is the target gene of mi R-451.In order to explore the effect of cadmium on head and kidney lymphocytes of common carp,on the basis of establishing the model of head and kidney lymphocytes of common carp exposed to cadmium,the models of mi R-451 knock-down / overexpression and mi R-451 and cacna1 ab co-knockdown cells were established respectively.The damage of carp head and kidney lymphocytes was analyzed by immunofluorescence staining,cellular calcium staining and MDC staining(autophagy staining).The expression of genes related to autophagy classical pathway was detected by q RT-PCR,Western Blot method,in order to clarify the mechanism of autophagy induced by targeting cacna1 ab in carp head kidney lymphocytes during cadmium exposure.The specific test results are as follows:1.In the cadmium-exposed carp head-kidney lymphocyte model,the m RNA expression of mi R-451 decreased and the m RNA expression of cacna1 ab increased(p < 0.05).The results of calcium staining showed that the lymphocytes of cadmium group were overloaded(p < 0.05).Both immunofluorescence staining and MDC staining showed the occurrence of autophagy of lymphocytes in cadmium group.At the same time,q RT-PCR results showed that the expression of ATG5,LC3-II and Beclin1 increased,while the expression of m TOR,p62 and Bcl-2 decreased(p < 0.05),while Western Blot results showed that the expression of LC3-I,LC3-II and Beclin1 increased,while the expression of p62 and Bcl-2 decreased(p < 0.05).2.The target gene of mi R-451 was predicted to be cacna1 ab by bioinformatics software Targetscan,and cacna1 ab was also verified to be the target gene of mi R-451 by constructing a dual-luciferase reporter gene system.q RT-PCR results showed that the m RNA level of cacna1 ab was decreased in the mi R-451 overexpression group and cacna1 ab knockdown group,while it was significantly increased in the mi R-451 knockdown group(p < 0.05),again proving the targeting relationship between mi R-451 and cacna1ab.3.In the mi R-451 knockdown / overexpression cell model,the results of calcium staining showed that the lymphocytes in the mi R-451 knockdown group were overloaded(p < 0.05).The results of immunofluorescence staining and MDC staining showed that autophagy occurred in mi R-451 knockdown group.At the same time,q RT-PCR results showed that the expression of ATG5,LC3-II and Beclin-1 increased,while the expression of m TOR,p62 and Bcl-2 decreased in mi R-451 knockout group(p < 0.05),while the expression of LC3-I,LC3-II and Beclin-1 increased and p62 and Bcl-2 decreased in mi R-451 knockout group(p < 0.05).4.In the co-knockdown cell model of mi R-451 and cacna1 ab,cacna1ab knockout can reduce the calcium overload and autophagy activation induced by mi R-451 knockdown.The results showed that cadmium exposure regulated autophagy of carp head and kidney lymphocytes through mi R-451 targeting cacna1 ab.In summary,cadmium exposure destroys the calcium homeostasis of carp head and kidney lymphocytes and regulates lymphocyte autophagy through the mi R-451/cacna1 ab axis.This study revealed for the first time the mechanism of mi RNA-mediated autophagy in head and kidney lymphocytes of common carp exposed to cadmium,which enriches the immunotoxicological mechanism of cadmium in fish and provides a new theoretical basis for the immune regulation mechanism of organisms. |
