| Objective:Chromatin remodeling INO80 complex can participate in cell differentiation through transcriptional regulation and DNA repair,but the specific mechanism is still unclear.In this study,we used river crab,Eriocheir sinensis,mouse spermatozoa,mouse liver,and15 d hepatocellular carcinoma(HCC)tissues as samples to analyze the regulation of mi RNA on chromatin remodeling protein expression during cell differentiation,to explore the role of INO80 complex subunit D The localization and the possible role of INO80D/Ino80d(written in mice after the slash,hereinafter)in cell differentiation were investigated.Methods:(1)Tissues from the testes of river crabs and mice were selected as the materials,and the differential expression of INO80D/Ino80 d in the spermatozoa of river crabs and mice at the infantile and mature stages were analyzed by relevant experimental techniques such as high-throughput sequencing,real-time fluorescence quantitative PCR(RT-q PCR),western blot(WB)and rabbit immunofluorescence(immunofluorescence,IF).(2)After left axillary subcutaneous implantation of mouse hepatocellular carcinoma cells(H22)in SPF grade mice,15 d growing HCC tissues and normal mouse liver tissues were taken as materials,and the expression and localization of Ino80 D were analyzed by RT-q PCR,WB and IF techniques.And the mi RNA targeting INO80 D was predicted using Target Scan,Miranda,and RNAwalk databases,and the expression of mi RNA was interfered with by RNA oligo to observe the changes of m RNA and protein level expression of its target gene Ino80 d,and further verify its possible targeting regulatory relationship and the regulatory mechanism of Ino80 d expression.Results:(1)INO80D/Ino80 d,a putative regulatory component of the INO80 chromatin remodeling complex,was differentially expressed in spermatophore tissues of adult and juvenile mice and river crabs(n=3,P<0.05),which could suggest that INO80D/Ino80 d may be involved in spermatophore development and spermatogenesis.(2)The expression pattern of INO80D/Ino80 d in river crab and mouse spermatozoa tissues was consistent(m RNA and protein expression levels of INO80D/Ino80 d were upregulated in adult crab and adult mouse spermatozoa tissues relative to juvenile crab and mouse spermatozoa tissues)outside.m RNA level expression of INO80D/Ino80 d was consistent with protein level expression.(3)INO80D/Ino80 d was mainly expressed in the nucleus of germ cells in river crab and mouse spermatozoa,but its expression differed in different germ cells of the two species: in river crab spermatozoa,the expression of INO80 D was significantly higher in spermatogonia and spermatocytes than in other cells,and its expression gradually decreased with cell differentiation,especially in spermatozoa where the expression of INO80 D was much lower.The expression of INO80 D in spermatozoa was very low.However,in mouse testis tissues,the expression of Ino80 d protein was significantly higher in spermatogonia and spermatocytes than in other cells,especially in primary spermatocytes,secondary spermatocytes,and spermatozoa,and its expression was lower.(4)Prediction and analysis of mi RNA of INO80D/Ino80 d in river crab and mouse species:(1)In river crab spermathecae,mi R-92a＿10 was determined to be the mi RNA targeting INO80 D.RT-q PCR showed that the relative expression of mi R-92a＿10 in the spermathecae tissue of river crabs was(1.24 ± 0.77)lower in adult crabs than in juvenile crabs(The relative expression of INO80 D m RNA in adult crabs(8.42 ± 2.89)was significantly higher than that in juvenile crabs(1.17 ± 0.65),and the difference was statistically significant(n=3,P<0.05).suggesting that mi R-92a＿10 may have a negative regulatory relationship with INO80 D in river crab spermatozoa tissue.(2)In mice,mi R-92a-3p was identified as the mi RNA targeting Ino80 d.Bio Edit sequence comparison showed that this mi RNA had high sequence similarity(more than 90% base identity)with the mi RNA targeting the same gene(as mi R-92a＿10)in river crabs.RT-q PCR experiments showed that mi R-92a-3p relative expression in adult mouse testis tissue was(0.50 ± 0.09),which was lower than that in juvenile mouse testis tissue(1 ± 0.11);however,the relative expression of its target gene Ino80 d m RNA in adult mouse testis tissue was(1.82± 0.31),which was higher than that in juvenile mouse testis tissue(1 ± 0.16),and the difference was statistically significant(n=3,P<0.05).It was shown that mi R-92a-3p and Ino80 d may have a target-regulatory relationship in adult and juvenile mouse testis tissues.Creating mi R-92a-3p RNA oligo transfected mouse spermatogonia,RT-q PCR and WB results revealed that the expression of mi R-92a-3p was significantly up-regulated and Ino80 d m RNA and protein levels were down-regulated after transfection,which more deeply put indicates that mi R-92a-3p and Ino80 d may have a negative regulatory relationship.(5)The m RNA and protein expression levels of Ino80 d were significantly decreased in 15 d tumor tissues and normal mouse liver tissues,and the differences were statistically significant(n=3,P<0.05).The expression of Ino80 d at the m RNA level was consistent with that at the protein level.ino80 d was mainly localized in the nucleus of cells.In mouse HCC,mi R-92a-3p was identified as the mi RNA targeting Ino80 d.RT-q PCR showed that they were both differentially expressed in mouse normal liver tissue and 15 d tumor tissue(n=3,P<0.05).mi R-92a-3p was expressed in 15 d tumor tissue(39.33 ± 11.39),which was higher than that in mouse normal liver tissue(1.01 ± 0.14);however,its expression in 15 d tumor tissue was higher than that in mouse normal liver tissue(1.01 ± 0.14).The relative expression of Ino80 d m RNA in 15 d tumor tissues was(0.08 ± 0.03),which was lower than that in normal mouse liver tissues(1.08 ± 0.47).It indicates that mi R-92a-3p is also most likely to have a targeted regulatory relationship with the target gene Ino80 d.The mi R-92a-3p RNA oligo was created to transfect mouse H22 cells,and the results of RT-q PCR and WB revealed that after transfection,the expression of mi R-92a-3p was significantly down-regulated and Ino80 d m RNA and encoded protein were significantly up-regulated,which more deeply indicated that mi R-92a-3p might have a negative regulatory relationship with Ino80 d.Conclusions:(1)Candidate INO80D/Ino80 d in river crab spermathecae and mouse spermathecae may play an important role in spermathecae development and spermatocyte differentiation by participating in biological processes such as chromatin remodeling,cellular response to DNA damage stimuli,and regulation of chromosome organization involved in nucleosome sliding;mi RNA of candidate chromatin remodeling complexes,mi R-92a＿10/mi R-92a-3p(the corresponding mi RNA in mice after the slash,same below)play a role in the development of spermatophore and differentiation of spermatocytes in river crab and mice through the regulation of INO80D/Ino80 d,respectively,although the targeting and regulation relationship between mi R-92a＿10/mi R-92a-3p and INO80D/Ino80 d needs to be verified by relevant experiments.(2)Ino80d may play an important role in hepatocyte dedifferentiation carcinogenesis by participating in the cellular response to DNA damage stimuli and chromosomal organization regulation in cancer chromatin remodeling together with other INO80 chromatin remodeling complex subunits during mouse HCC.mi R-92a-3p plays a role in mouse HCC development by regulating Ino80 d,but their targeting regulation needs to be further validated.their targeted regulatory relationship needs to be deeply verified by relevant experiments. |
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