Identification, Verification And Functional Analysis Of Differentially Expressed Genes In Hyperthermia Induced Neural Tube Defects Of Golden Hamster

Formation of neural tube is an important event during early embryogenesis and involves formation of neural plate, neural plate folding into neural ridge and neural ridge merging into neural tube. Neural tube is the primordium of central nervous system (CNS). The genesis and differentiation of neural tube are precondition of normal development of brain and spinal cord. During neural tube development, many environmental teratogens may have adverse effects and thus induce neural tube defects (NTDs). NTDs are amongst the most prevalent and serious of all human congenital anomalies and place an enormous emotional and economic burden on the population.Since last century, scientists have been working on exploring various environmental teratogens that may induce NTDs. They found that hyperthermia is a prevalent and unavoidable factor. This has motivated extensive research about mechanism of hyperthermia induced NTDs. There is now an extensive literature about the effects of hyperthermia on cell proliferation, apoptosis, differentiation, migration, adhesion and aggregation as well as on tissue induction and organogenesis. Previous studies revealed that many genes participate in the course of hyperthermia induced NTDs. None or insufficent expression of some genes and specific or excessive expression of some other genes may induce NTDs. Hyperthermia induced NTDs are complicated because genes including both upregulated and downregulated are involved. However, there is still no detailed research about the differentially expressed genes in hyperthermia induced NTDs.Recently, several PCR-based techniques for differential genes screening have been established. A technique named suppression subtractive hybridization (SSH), owing to its high specificity and parity, has been widely applied to many molecular cloning studies for identification of differentially expressed genes in disease, development and different tissues. Here, we constructed subtractive cDNA libraries of hyperthermia induced NTDs of golden hamster in two directions using SSH. Then we identified both upregulated and downregulated genes in hyperthermia induced NTDs from these two subtractive cDNA libraries. Sequence and homology analysis were made to these differentially expressed genes and Northern blot were performed to verify differential expression levels of these genes. Then we selected Npm1, an important gene for embryogenesis as our study target and explored its function in vitro.PART ONE Construction and Validation of Forward and Reverse Subtractive cDNA Libraries of Hyperthermia Induced NTDs of Golden HamsterTo identify differentially expressed genes in hyperthermia induced NTDs of golden hamster, we first constructed subtractive cDNA libraries in two directions. Total RNA were isolated from both control and hyperthermia groups and then were reverse transcripted into double-strand cDNA (ds cDNA) using SMART^TM cDNA synthesis method. We applied method of phenol and chloroform extraction to purify ds cDNA. Then, the purified cDNA were digested by RsaI to generate shorter, blunt-ended ds cDNA fragments and purified again. The final concentration of cDNA were adjusted to 300 ng/μl. Then SSH was carried out in two directions: forward and reverse, with PCR-Select cDNA Subtraction Kit. For the forward SSH, ds cDNA derived from control group was considered the 'driver' pool, ds cDNA derived from hyperthermia group the 'tester' pool. From the forward SSH, we could identify upregulated genes in hyperthermia induced NTDs. For the reverse SSH, ds cDNA from hyperthermia group was regarded as the 'driver' pool, ds cDNA from control group the 'tester' pool. From the reverse SSH, we could identify downregulated genes in hyperthermia induced NTDs. The housekeeping gene—Gapdh was used as control in SSH to evaluate subtractive efficiency. The significant decrease of Gapdh after SSH suggested high subtractive efficiency in our experiment. Then the purifed subtracted cDNA were ligated with pGEM-T Easy vector and transformed into E. Coli DH5αusing CaCl₂ method. The two subtractive cDNA libraries were separately plated on LB agar plates containing ampicillin (100μg/ml) and IPTG/X-gal. The results of blue-white selection and clony PCR showed that the two subtractive cDNA libraries included differentially expressed gene fragments with length from 150 bp to 1 kb. This suggests that our subtractive cDNA libraries were successfully constructed and suitable for identification of differentially expressed genes in hyperthermia induced NTDs.PART TWO Screening, Sequencing and Verification of Differentially Expressed Genes in Hyperthermia Induced NTDs of Golden HamsterGenerally, a subtractive cDNA library contains so many differentially expressed genes that researchers cannot sequence them all. So we just selected clones from the two subtractive cDNA libraries randomly. Then we sequenced these clones and compared the sequencing results with Genbank database by blastn software. From the reverse subtractive cDNA library, we identified 14 downregulated genes in all and found them all homologous to known genes with high homology. The proteins encoded by these genes included ribosomal proteins, metabolic enzymes, transcription and translation factors and others. Northern blot showed that expression of all these 14 genes decreased significantly in defected neural tubes induced by hyperthermia. However, from the forward subtractive cDNA library, most of the inserts were short and only two meaningful fragments were identified after sequence and homology analysis. The two inserts were of the same sequence and homologous to mouse Pgk1 with homology of 92%. The increased expression of Pgk1 in defected neural tubes induced by hyperthermia was also confirmed by Northern blot. In this part, we verified these differentially expressed genes by Northern blot, showed differential expression of these genes are strongly related to hyperthermia induced NTDs. PART THREE Functional Study of Npm1 in vitro Npm1 was a gene identified from the reverse subtractive cDNA library. Previous studies showed that Npm1 plays an important role in embryogenesis. Npm1^-/- embryos were consistently smaller in size compared with normal ones. The Npm1^-/- mutants also showed distinct developmental abnormalities. Most noticeably, they displayed deficient anterior brain organogenesis, with complete absence of eyes. This suggested that Npm1 is essential for neurogenesis. So we selected Npm1 to explore its role in NTDs induced by hyperthermia. Neural stem cells (NSCs) were used as our study model in vitro. As Npm1 was downregulated in hyperthermia induced NTDs, we suppressed expression of Npm1 in NSCs by RNA interference (RNAi). After RNAi, Npm1 was downregulated at both mRNA and protein levels. Then we studied cell proliferation, apoptosis and differentiation in NSCs after Npm1 suppression. As a result, suppression of Npm1 decreased proliferation rate of NSCs, increased apoptosis occurrence but did not seem to make any significant difference in cell differentiation. As there was an increase of p53 protein after Npm1 was suppressed, we speculated that apoptosis of NSCs induced by downregulation of Npm1 was strongly related to p53 pathway.In our work, we successfully constructed forward and reverse subtractive cDNA libraries of hyperthermia induced NTDs of golden hamster. And we identified several downregulated and upregulated genes involved in hyperthermia induced NTDs. In addition, we employed RNAi with NSCs in vitro to study the function of Npm1 in hyperthermia induced NTDs. Our work provides important theoretical and technical support for mechanism and prevention of hyperthermia induced NTDs.