The Study On The MRNA/lncRNA Expression Profile In Human Cumulus Cells Related To Early Embryo Development

Part Ⅰ The Study on the m RNA Expression Profile in Human Cumulus Cells Related to Early Embryo DevelopmentObjective To detect the m RNA expression profile of human cumulus cells related to early embryo development, and analyse and confirm the differential expressed genes.Methods（1） Cumulus cells were collected from women who experienced IVF-ET and was grouped according to embryo quality. The RNA expression levels in six specimens were tested in order to comply with the requirements of the chip experiment. The RNA in the specimens were marked with Cy3 fluorescent dyes. The RNA expression levels in cumulus cells specimens were tested with Arraystar Human m RNA V2.0 expression spectrum chips（8x60K, Arraystar）. The microarray image information was collected, standardized and evaluated.（2） Differential expressed m RNAs were screened with two classification difference methods and showed with volcano plots and hierarchical cluster diagram. The enrichment method was employed as further analysis of the differential expressed m RNAs. Gene ontological analysis and KEGG database was used to analyze the screened differential genes.（3） Real-time quantitative PCT was used to screen related m RANs, select and verify the target gene.Results（1） Six samples of cumulus cells that were collected from women treated with IVF-ET met with the requirement of m RNA expression spectrum chip. The RNA OD260/280 ratio in the group A were 1.95, 1.92 and 2.04 respectively. The RNA OD260/280 ratio in the group B were 2.05, 2.00 and 1.94 respectively. Two groups ofsamples all met the requirements of the chip. The samples of tag activity for specificity（pmol/ml dye）/（ mg/ml c RNA） of group A were 21.42, 21.70 and 20.64 respectively and those of group B were 21.25, 21.47 and 21.00 respectively. All of them met with the experiment requirement of chip. A total of 15797 m RNAs target genes were detected in two groups. After the filtered data quaility assessment, the box-plot analyzed that the m RNA expression of six samples was close to a level and scatter diagram analysis showed that there were a large of differentially expressed m RNAs in the two groups of cumulus cells.（2） Screening analysis of differentially expressed m RNAs showed that there were 810 differentially expressed genes. The number of up-regulated expressed genes was 91 in group B vs group A and those of down-regulated expressed genes was 719. Gene ontology analysis showed that there were 112 genes participating in biological processes, 24 genes paticipating in cell components and 22 genes participating in cellular functions among the up-regulated expressed m RNAs genes, and 389 genes participating in biological processes, 94 genes paticipating in cell components and 85 genes participating in cellular functions among the down-regulated expressed m RNAs genes. Signal pathway analysis showed that the up-regulated differentially expressed genes related to 5 signal pathways and down-regulated differentially expressed genes related to 12 signal pathways.（3） By selecting some of target gene and verifying it with real-time quantitative PCR showed that the result of q PCR was consistent with that of chips.Conclusions There were a large differentially expressed m RNAs between two groups. Gene ontology analysis and signal pathway analysis indicated that these differentially expressed m RNAs were involved in various biological pathways and signaling pathways of oocyte developmental potential. These differentially expressed m RNAs genes possiblely participate in the oocyte development and early embryo development by transcription or translation lead to the change of proteins.Part Ⅱ The Study on the Lnc RNA Expression Profile in Human Cumulus Cells Related to Early Embryo Development and the Correlation Analysis between Lnc RNAs and m RNAsObjective To detect the lnc RNA expression profile of human cumulus cells related to embryo developmental potential, and analyze and confirm the differential expressed genes.Methods（1） Cumulus cells were collected from women who experienced IVF-ET and was grouped according to embryo quality. The RNA expression levels in six specimens were tested in order to meet with the requirements of the chip experiment. The RNA in the specimens were marked with Cy3 fluorescent dyes. The RNA expression levels in cumulus cells specimens were tested with Arraystar Human lnc RNA V2.0 expression spectrum chips（8x60K, Arraystar）. The microarray image information was collected, standardized and evaluated.（2） Differential expressed lnc RNAs were screened with two classification difference methods and showed with volcano plots and hierarchical cluster diagram. The enrichment method was employed as further analysis of the differential expressed lnc RNAs. The differentially expressed lnc RNAs were classified and that of the subgroups also were analyzed.（3） Real-time quantitative PCR was used to screen related lnc RANs, select and verify the target lnc RNAs. According to the chip data of differentially expressed m RNAs and lnc RNAs, we try to find out the common expressed genes. Based on relationship of cumulus cells and early embryo development, coding-non-coding gene co-expression network analysis was established between the differentially expressed m RNAs and lnc RNAs in order to construct gene regulatory networks.Results（1） Six samples of cumulus cells that were collected from women treated with IVF-ET to meet the requirement of lnc RNA expression spectrum chip. The RNA OD260/280 ratio in group A were 1.95, 1.92 and 2.04 respectively. The RNA OD260/280 ratio in group B were 2.05, 2.00 and 1.94 respectively. Two groups of samples conformed to the requirements of the chip. The samples of tag activity for specificity（pmol/ml dye）/（mg/ml c RNA） of group A were 21.42, 21.70 and 20.64 respectively and those of group B were 21.25, 21.47 and 21.00 respectively. All of them met with the experiment requirement of chip. A total of 20563 lnc RNAs were detected in two groups. After the filtered data quaility assessment, the box-plot analyzed that the lnc RNA expression of six samples was close to a level and scatter diagram analysis showed that there were a large of differentially expressed lnc RNAs in the two groups of cumulus cells.（2） Screening analysis of differentially expressed lnc RNAs showed that there were 633 differentially expressed lnc RNAs. The number of up-regulated expressed lnc RNAs was 124 in group B vs group A and those of down-regulated expressed lnc RNAs was 509. Lnc RNAs classification and subgroup analysis showed that there were 925 lnc RANs with enhancer-like function and 166 enhancer near encoding genes, 2041 linc RNAs and 373 encoding genes, and 387 lnc RNAs with HOX cluster sequences.（3） By selecting some of lnc RNAs and verifying it with real-time quantitative PCR showed that the result of q PCR was consistent with that of chips. Based on the chip data, there were three pairs m RNAs and lnc RNAs, which have common expression of genes, ASNS, CALB2 and NEK7, corresponding lnc RNAs were ENST00000429197, NR₀27910 and Y00062. Three pairs of them were down-regulated in group B vs group A.Conclusions（1） There were a large differentially expressed lnc RNAs between two groups. The results indicated that the differentially expressed lnc RNAs in human cumulus cells possibly participate in the regulatory mechanism of early embryodevelopment. Lnc RNAs subgroups analysis suggested that there were a lot of different functions of lnc RNAs related to transcription regulation of m RNAs, epigenetic modification and some regulation role.（2） The results of lnc RNA-m RNA correlation analysis showed that ASNS, CALB2 and NEK7 and corresponding lnc RNAs ENST00000429197, NR₀27910 and Y00062 possibly have correlation in human cumulus cells regulation mechanism of early embryo development.