ZHU Rui Qing, SONG Le Quan, JIANG Lin, LIU Yu, ZHAO Li, WANG Hao Yu, ZHANG Jing,XU Xin Ping, DONG Ji, YAO Bin Wei, ZHAO Xue Long, WANG Hui,#,SHI Xu Liang, and PENG Rui Yun,#

Microwave-related devices are commonly used.A certain dose of microwave might induce cognitive injuries[1]. The frequency of mobile 4G communication and wireless networks is located in the L-band and C-band range, respectively. We found that the accumulative exposure group,exposed to 1.5 GHz and 4.3 GHz microwaves, was more severely damaged than the single exposure group, indicated by prolonged average escape latency and structural damage to the hippocampus[2].

The proteomic analysis of the hippocampus in the accumulative microwave exposure was discussed in our previous work[3]. However, wholetranscriptome sequencing, especially of messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs),for identifying sensitive markers of microwaveinduced brain changes has not been reported.

Sixteen male Wistar rats 200 ± 20 g were divided into four groups. The groups, microwave exposure methods, and specific absorption rate (SAR) values are shown in Supplementary Table S1 (available in www.besjournal.com). All experiments were designed and reported according to the Animal Research: Reporting of In Vivo Experiments (ARRIVE)guidelines. Details of microwave exposure systems have been given in previous studies[4].

At 6 h after accumulative exposure, hippocampal samples were extracted, and transcriptome sequencing was performed following the recommended protocol. The remaining RNA of each sample after sequencing was used for qRT-PCR.Supplementary Table S2 (available in www.besjournal.com) shows the primer sequences used in the qRT-PCR.

Data were presented as mean and standard deviation. The data were subjected to two-way analysis variance using the software SPSS version 19.Differences atP＜ 0.05 were considered to be significant.

The mRNAs exhibiting significant differences in expression between the two groups were counted and are shown in Supplementary Figure S1 (available in www.besjournal.com). Compared to the S group,714 mRNAs were upregulated, and 1,069 mRNAs were downregulated in the L10 group. In the C10 group, 1,119 mRNAs were upregulated, and 2,076 were downregulated. In the LC10 group, 923 mRNAs were upregulated, and 2,129 were downregulated.

Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyzes were performed to search for possible biological processes related to cognitive changes. Following the GO functional enrichment analysis, the distribution of differentially expressed mRNAs in various biological processes, cellular components, and molecular functions was determined in the exposure groups relative to the S group. For the biological processes,differentially expressed mRNAs in the three exposure groups were mainly associated with positive regulation of transcription by RNA polymerase II, negative regulation of transcription by RNA polymerase II, transmembrane transport, ion transport, and brain development (Figure 1A, 1B,and 1C). Differential expression of mRNAs induced by microwave exposure was mainly located in the membrane, nucleus, cytoplasm, and mitochondrion(Figure 1A, 1B, and 1C). The molecular functions of differentially expressed mRNAs included nucleic acid binding, protein binding, and ion channel activity(Figure 1A, 1B, and 1C). KEGG pathway analysis suggested that glutamatergic synapse, mitogenactivated protein kinase signaling pathway, cAMP signaling pathway, and RNA degradation were involved in the differential expression of mRNAs in the S group and exposure groups (Figure 2A, 2B, and 2C). The biological processes, cellular components,molecular functions, and KEGG pathways between the LC10 group and L10 group are shown in Figures 1D and 2D. Figures 1E and 2E show the GO analysis and KEGG pathways of differentially expressed mRNAs between the LC10 and C10 groups. The differentially expressed mRNAs between the L10 and C10 groups were analyzed and are shown in Figures 1F and 2F.

Relative to the S group, more than 1,400 differentially expressed lncRNAs were found in each exposure group (Supplementary Figure S2 available in www.besjournal.com).

Relative to the S group, there were two predicted target genes of differentially expressed lncRNAs in the L10 group connected with cognitive functions. The biological processes mainly included synaptic plasticity and calcium ion transport. The cellular components were located in the synapse and the membrane. Molecular functions focused on syntaxin binding and calcium-dependent protein binding (Supplementary Table S3 available in www.besjournal.com).

The differentially expressed target mRNAs and lncRNAs in the C10 group were analyzed relative to those of the S group. The biological processes included brain development and tubulin complex assembly. The cellular components were located in the SNARE complex and synapse. Molecular functions focused on SNARE binding, syntaxin binding, and tubulin binding (Supplementary Table S3).

The differentially expressed target mRNAs and lncRNAs in the LC10 were compared with those of the S group. The biological processes focused on synaptic plasticity and calcium ion import. The cellular components were located in synapses and membranes. The molecular functions included calcium channel activity and GTPase activity(Supplementary Table S3).

When comparing the LC10 group with the L10 group or the C10 group, there were no cognitively connected differentially expressed lncRNAs and targeted mRNAs. When comparing the L10 group with the C10 group, only one cognitively connected differentially expressed lncRNAs was found(Supplementary Table S3). The KEGG pathways of the differentially expressed lncRNAs connected to the cognitive functions in the exposure groups mainly included the synaptic vesicle cycle and glutamatergic synapse (Supplementary Table S4 available in www. besjournal.com).

Figure 1. GO analysis of differentially expressed mRNAs in the sham and microwave exposure groups.A: biological processes (BP), cellular components (CC), and molecular functions (MF) in L10 vs. S;B: biological processes, cellular components, and molecular functions in C10 vs. S; C: biological processes,cellular components, and molecular functions in LC10 vs. S; D: biological processes, cellular components,and molecular functions in LC10 vs. L10; E: biological processes, cellular components, and molecular functions in LC10 vs. C10; F: biological processes, cellular components, and molecular functions in L10 vs.C10.

From combined GO enrichment and KEGG pathway analyzes, five-pair genes were selected for the following validation related to the cognitive changes. The genes, their transcripts, and the fold difference between groups are shown in Supplementary Table S5 (available in www.besjournal.com). The qRT-PCR was used to verify the sequencing results. Compared with that of the S group, the expression level of lncRNA (MSTRG.1068.1)decreased only in the C10 group, but the level of expression of mRNA (ENSRNOT00000002044) did not significantly change, which was not consistent with the sequencing data (Figure 3A). Compared with that of the S group, the level of expression of lncRNA (MSTRG.27033.1) increased significantly in the C10 and LC10 groups, and the level of expression of mRNA(ENSRNOT00000083373) decreased in the L10, C10,and LC10 groups, which was consistent with the sequencing data (Figure 3B). The lncRNA(MSTRG.31953.1) was targeted with two mRNAs(ENSRNT00000023196 and ENSRNOT000000023066).The qRT-PCR results of lncRNA (MSTRG.31953.1) and mRNAs (ENSRNT00000023196 and ENSRNOT 000000023066) showed a decrease in their expression in the L10, C10, and LC10 groups, which was consistent with the sequencing data (Figure 3C). Only mRNA (ENSRNOT00000022351) decreased in the LC10 group. The lncRNA (MSTRG. 35922.1) and mRNA(ENSRNOT00000022351) validation results were not consistent with the sequencing results (Figure 3D).Statistical analysis results are shown in Supplementary Table S6 (available in www.besjournal.com). The results indicated that the lncRNAs (Slc24a2 and Pdgfb)and mRNAs (Slc24a2, Pdgfb, and Syngr1) might be sensitive genes of microwave exposure, which should be considered for future study.

Figure 2. KEGG pathway analysis of differentially expressed mRNAs in the sham and microwave exposure groups. A: KEGG pathways in L10 vs. S; B: KEGG pathways in C10 vs. S; C: KEGG pathways in LC10 vs. S;D: KEGG pathways in LC10 vs. L10; E: KEGG pathways in LC10 vs. C10; F: KEGG pathways in L10 vs. C10.

Recently, lncRNAs have attracted much attention due to their roles in transcriptional, posttranscriptional, and epigenetic networks and in certain physiological and pathological processes.Many lncRNAs are expressed in the central nervous system and play an important role in regulating neural functions such as central nervous system development, synaptic plasticity, and stress response. Changes in the expression levels of specific lncRNAs have been reported[5]. Through the mRNA and lncRNA analyzes, we found that brain development might be the main biological process.The glutamatergic synapse and synaptic vesicle cycle should be considered important pathways for microwave radiation studies. Previous studies have also indicated the important roles of glutamate and glutamatergic synapse in microwave-induced hippocampal injuries[6].

Slc24a2, also named NCKX2, plays a role in the calcium ion transmembrane transport[7]. We have previously reported calcium efflux during microwave exposure in primary hippocampal neurons[8]. The NCKX is another possible reason for microwaveinduced calcium changes, which should be given potential attention in future studies.

The lncRNA Pdgfb was targeted to two mRNAs(Pdgfb and Syngr1). The mRNA of Pdgfb plays an essential role in the regulation of embryonic development and cell proliferation. The mRNA of Syngr1 probably plays a role in synaptic-like microvesicle formation and/or maturation[9]. An abnormal number of synaptic vesicles were found in transmission electron microscope pictures of the hippocampus after microwave exposure[10].

Figure 3. Validation data of the selected genes using qRT-PCR. A: qRT-PCR results of lncRNA(MSTRG.1068.1) and its target mRNA; B: qRT-PCR results of lncRNA (MSTRG.27033.1) and its target mRNA; C: qRT-PCR results of lncRNA (MSTRG.31953.1) and its target mRNA; D: qRT-PCR results of lncRNA(MSTRG.35922.1) and its target mRNA. Compared with the S group, *P ＜ 0.05 and **P ＜ 0.01.

In conclusion, exposure to microwaves might affect the biological processes of brain development and ion transport, KEGG pathways of the glutamatergic synapse, and synaptic vesicle cycle,thereby inducing neurological impairment. Exposure to microwaves causes differential expression of lncRNAs (Slc24a2 and Pdgfb) and mRNAs (Slc24a2,Pdgfb, and Syngr1). The response of differentially expressed RNAs in body fluid samples should be further explored.

&These authors contributed equally to this work.

#Correspondence should be addressed to WANG Hui,E-mail: wanghui597bj@163.com; SHI Xu Liang, E-mail:shixl163@163.com; PENG Rui Yun, E-mail:ruiyunpeng18@126.com Tel/Fax: 86-10-66931236

Biographical notes of the first authors: ZHU Rui Qing,male, born in 1991, MA, majoring in neurobiological damage of electromagnetic radiation; SONG Le Quan,male, born in 1997, PhD, majoring in neurobiological damage of electromagnetic radiation; JIANG Lin, female,born in 1998, MA, majoring in electromagnetic radiation psychology and cognition.

Received: June 16, 2022;

Accepted: September 26, 2022