Correlation and Mechanism Study of Multivoxel 1H-MRS Parameters, Cho / Cr and CHKA

Background Glioma is a common malignant brain tumor with strong invasiveness and poor prognosis. in order to improve the survival rate of patients, there is an urgent need to nd the targeted molecular mechanism affecting its invasion and development. Methods In this study, the differential regions of Cho/Cr metabolism of tumor cells were identied by multi-voxel 1 H-MRS and the tumor tissues of Cho/Cr high metabolism group and Cho/Cr low metabolism group were accurately obtained by combining neuronavigation system. The expression of CHKA in different Cho/Cr metabolic groups was veried by Real-timePCR and Western-blot. Then we used CHKA inhibitor MN58b and short hairpin RNA to study the role of CHKA in the proliferation and invasion of U251 glioma cells, and the relationship between CHKA and EMT in nude mice. Results We found that the expression of CHKA in Cho/Cr high metabolism group was signicantly higher than that in Cho/Cr low metabolism group. Decreased CHKA activity can inhibit the proliferation and invasion of glioma cells and slow down the process of EMT. Conclusion CHKA may be an oncogenic gene, which affects the invasion and development of glioma cells by regulating the process of tumor EMT, and is expected to become a molecular target for glioma therapy, and multi-voxel 1 H-MRS parameter Cho/Cr may become an important clinical index for detecting CHKA expression.


Introduction
Glioma is the most common primary central nervous system tumor, accounting for 40% -50% of intracranial tumors. The average survival time after diagnosis is less than 15 months [1,2]. Because of the strong invasive nature of glioma [3] and extensive in ltration during the growth process it cannot be completely removed by surgery, and the recurrence rate is high [4]. Therefore, on the premise of protecting normal brain tissue, striving to remove tumor tissue to the greatest extent is the fundamental purpose of glioma treatment. Although MRI is a common method for the diagnosis of brain tumors in the central nervous system, it cannot evaluate the changes of metabolites around the whole lesion tissue, and it also has some limitations in judging the pathological grade and in ltration range of glioma. However, 1 H-MRS can be used to monitor the changes of metabolites around tumor tissue in vivo [5]. Quantitative and qualitative analysis of some speci c areas is carried out by using magnetic resonance imaging and chemical shift, and the disease is evaluated and diagnosed according to the quantitative changes, which improves MRI at the molecular level. The diagnostic value of MRI for brain tumors can be used as a powerful supplement to conventional MRI detection methods [6]. NAA, CR, Cho, LAC, and LIP were detected by multivoxel 1 H-MRS [7,8], Among them, Cr peak is located at 3.02ppm, which is the second highest peak of normal brain wave spectrum. It is composed of γ -aminobutyric acid, creatine, phosphocreatine, lysine and glutathione. Its total amount is relatively stable among different tissues of the same individual, so it is often used as a reference value to measure the content of other metabolites. Cho peak is located at 3.2ppm, which is one of the main components of phospholipid metabolism in brain [9]. Its main components include PCho, PC and GPC. The increase of Cho peak can infer that the phospholipid metabolism of cell membrane is accelerated and the cell division and proliferation are vigorous.
Choline kinase is a key enzyme in Kennedy's Choline metabolic pathway [10]. It is encoded by two genes, CHKA and CHKB, through homodimer or heterodimer. In vivo [11] CHKB can only play the role of ethanolamine kinase, while CHKA has the dual activity of Choline kinase and ethanolamine kinase. CHKA with Choline kinase activity can phosphorylate free Choline to Choline phosphate. PhosphatidylCholine is an important second messenger molecule, which plays a key role in DNA synthesis and promotes cell proliferation. Thus, CHKA can indirectly participate in cell mitosis through Choline phosphate and affect the physiological activities of cells. In recent years, some studies have also con rmed that [12][13][14] overexpression of CHKA can induce malignant transformation of some normal cells, and silencing or using inhibitors can inhibit these malignant biological behaviors.
EMT refers to the biological process in which epithelial cells lose their epithelial phenotypic characteristics and transform into broblast-like stromal phenotypes [15]. EMT plays an important role in the invasion and migration of malignant tumor cells. The expression of epithelial cell adhesion protein Ecadherin is decreased, while the expression of mesenchymal cell characteristic proteins Vimentin and Ncadherin is up-regulated, which leads to the weakening of intercellular polarity and adhesion ability and the enhancement of tumor cell invasion and migration ability. Although in recent years, more and more studies have found that EMT is closely related to the occurrence and development of tumors of various epithelial sources, such as lung cancer, breast cancer, liver cancer and so on, the role and speci c molecular mechanism of EMT in brain gliomas are still unclear.
In multivoxel 1 H-MRS studies, Cho / Cr is commonly used to re ect the level of Cho metabolism. The increase of Cho / Cr is correlated with the grade of glioma. [16,17] Cho / Cr guiding the resection range of glioma can de nitely improve the therapeutic effect and prognosis [18]. Whether CHKA, as a key enzyme in Choline metabolism is involved in the occurrence and development of glioma, and whether it is related to multivoxel 1 H-MRS parameters, to verify these conjectures, this study rst veri ed the correlation between the multivoxel 1 H-MRS parameter Cho / Cr and the expression of CHKA from clinical samples with different Cho / Cr metabolism. Then, we used lentiviral transfection to down regulate the expression of CHKA in U251 glioma cells, and used MN58b, a CHKA inhibitor, to inhibit the biological activity.

Clinical samples
Nineteen patients with high-grade gliomas were selected from the Department of Neurosurgery of Ningxia Medical University General Hospital (March 2015 to November 2018, all con rmed by postoperative pathology). The patients included, 9 males and 10 females, aged 7-68 years, with an average age of 45.6 years. Before multivoxel 1 H-MRS examination, all patients did not receive radiotherapy and chemotherapy, and had no history of brain injury and/or brain surgery. This study has been reviewed by the research ethics committee of Ningxia Medical University, and informed consent forms were signed with patients or their families before operation.

Image processing
Using signa HDX 3.0T superconducting MRI system made by GE company in the United States, multi voxel 1 H-MRS was performed after conventional MRI plain scans and enhanced scans. MRI was used to determine the location of the lesion. The multi voxel 1 H-MRS image and structure image data were transmitted to the post-processing workstation through LAN. The multi voxel analysis is carried out for each layer by using the self-contained software, and a 3D chemical displacement imaging is generated and converted into gray value image. The image contains standardized Cho / Cr information, and the Cho / Cr level of each voxel is represented by pseudo color image. Select the highest and lowest points of Cho / Cr as the region of interest and save them in DICOM format. The saved MRS images were imported into 3D interface, reconstructed and saved in axial direction. After being imported into navigation workstation, MRS sequences and structural images were fused and reconstructed, and the reconstructed sequences were exported to cranial software. In the fusion interface, the conventional anatomical sequences and MRS sequences were combined and multivoxel 1 H-MRS was added. The images were accurately fused into the intraoperative neuro-navigation system. With the aid of neuro=navigation, the highest and lowest Cho / Cr levels were accurately selected, and the tumor tissues in these two regions were taken as tissue samples, which were Cho / Cr high metabolism group and Cho / Cr low metabolism group, respectively.

Quantitative real time PCR
Total RNA was extracted from tumor tissue samples of Cho / Cr high metabolism group and Cho / Cr low metabolism group by Omega Kit (Omega , USA), and SYBR PremixExTaq TM . Gamma kit (Takara , Japan)was used to reverse the total RNA into cDNA, and then real-time PCRwas performed. The target gene CHKA primer sequence: upstream 5 '-CGGAAAAGTGCTCCTGCGGCT-3', downstream 5 '-AACCAAAGCTGTGCAGCCAA-3'. β -actin was selected as the internal reference gene. Samples were added according to the 20 μ l PCR system in the instruction manual and tested on the computer.

Western-blot Testing
The protein from Cho/Cr high metabolic group and Cho/Cr low metabolic group was extracted by full protein extraction kit and quanti ed by BCA. SDS-PAGE gel electrophoresis was carried out on the 40 micron g/ pore size. After electrophoresis, the protein on the gel was transferred to PVDF membrane at 300mA and 45 minutes. PVDF membrane was placed in 5% skimmed milk powder and sealed at room temperature for 2 hours then, it was added into CHKA primary antibody (Abcam , USA) for overnight on a 4 ℃ shaking table. The next day, the PVDF membrane was washed 5 times with TBST for 5 minutes each time. The second antibody of the corresponding species was incubated for 2 hours. After cleaning 5 times with TBST, the ECL emitted light.

Cell culture and lentiviral transfection
U251 cells (Shanghai cell bank of Chinese Academy of Sciences) were taken out from liquid nitrogen and placed in a 37℃ constant temperature water bath for 3-5min for resuscitation. The cell suspension in the cryopreserved tube was inhaled into a centrifuge tube containing 3ml culture medium (GIBCO ,USA) in an ultra-clean bench. Centrifugation was performed at 1000 RMP for 5 min. the supernatant was discarded. The cells at the bottom of the tube were used with 10% fetal bovine serum (GIBCO, USA) The DMEM with 1% double antibody was resuspended and seeded into a culture bottle, and then cultured in a cell incubator containing 5% CO2 at 37℃. U251 cells in logarithmic growth phase were seeded in a 6-well plate at the density of 1×10 5 . After 24 hours, CHKA silencing lentivirus diluted with DMEM and its control lentivirus were added. After 12 hours of incubation, the DMEM medium containing 10% fetal bovine serum was replaced. After 48 hours of culture, the uorescence of transfected cells was observed by uorescence microscope. The cells were screened with puromycin (2 μ g / ml), and then identi ed by realtime PCR and Western blot.

Clonal activity was detected by clonal assay
The cells in logarithmic growth phase of CHKA group and vector group were digested and inoculated into a 6-well plate containing 2 ml medium at a density of 200 cells / well. The cells were evenly inoculated by shaking gently and then cultured in a cell incubator with 5% CO2 at 37℃ for 2-3 weeks. The cells were observed every 3 days. When cell clones were visible in the 6-well plate, the culture was terminated and the supernatant was discarded were raised in a SPF environment for one week. U251 cells suspended in serum-free medium ( 1x10 7 ) were injected into the axilla of the right forelimb of each mouse. When the volume of the tumor on the right side of the mouse reached about 100mm 3 , the mice were divided into two groups, 6 in each group.
The experimental group was intraperitoneally injected with MN58b (2mg/kg/ days), and the control group was injected with normal saline. After 14 days of continuous treatment, all nude mice were killed and samples were taken as required.

Immunohistochemistry
The para n-embedded tumor specimens were cut into 4μm thick slices, which were routinely dewaxed and placed in sodium citrate buffer for antigen repair, cooled naturally for 2 hours, incubated with 3% hydrogen peroxide solution at room temperature for 10 minutes, and sealed with goat serum. Removing the sealing solution, the primary antibody E-cadherin (Servicebio,1:300), N-cadherin (Servicebio,1:300) and Vimentin (Servicebio,1:200) were incubated overnight at 4 ℃, and the second antibody (Servicebio,1:500) was added the next day. After DAB staining, the expression of E-cadherin, N-cadherin and Vimentin in glioma was observed under microscope.
2.11Statistical analysis SPSS 24.0 software was used for statistical analysis. The data results were expressed as mean ± standard deviation ( ± s). t-Test was used for comparison of two samples, one-way ANOVA was used for diversity comparison, and SNK-q test was used for comparison between two samples. The difference was statistically signi cant (P<0.05).

Image ndings
Conventional MRI results showed that the left frontal lobe of glioma patients showed low signal on T1, high signal on T2, and high signal on T2 Flair. No obvious enhancement was found after conducting an enhancement. There were different degrees of peritumoral edema (Fig. 1A).
The results of multivoxel 1 H-MRS showed that compared with normal brain tissue, the peak of NAA was signi cantly decreased, the peak of Cho was signi cantly increased, and the peak of Cr was slightly decreased. The ratio of Cho / Cr in different regions of glioma was signi cantly different (Fig. 1B). The processed spectral images were accurately fused into the neuro-navigation workstation, and the tumor tissues at the highest and lowest points of Cho / Cr were accurately obtained intraoperatively with combined neuro-navigation (Fig. 1C).

The expression of Cho / Cr in multivoxel 1H-MRS was correlated with the expression of CHKA
Through real-time PCR and Western blot, we found that the expression of CHKA in the tumor tissues of Cho / Cr high metabolism group was signi cantly higher than that of Cho / Cr low metabolism group. PCR results showed that there was signi cant difference between Cho / Cr high metabolism group (40.77 ± 24.18) and Cho / Cr low metabolism group (0.58 ± 0.52) (P < 0.001, t = 6.44, Fig.2A). Western blot results showed that Cho / Cr high metabolism group (1.26 ± 0.14) and Cho / Cr low metabolism group (0.49 ± 0.13) had signi cant difference (P < 0.001, t = 8.92, Fig. 2B, C). It can be seen that the expression of Cho / Cr in multivoxel 1 H-MRS is correlated with the expression of CHKA. The higher the Cho / Cr metabolic value, the higher the expression of CHKA.

Down regulation of CHKA expression can inhibit the clonal ability of glioma cells
In order to verify the relationship between the expression of CHKA and the clonal activity of glioma cells, we performed the cloning experiment of U251 glioma cell line transfected with CHKA silencing lentivirus and its control cell lines (shCHKA group and vector group). The experimental results showed that there was signi cant difference between shCHKA group (25 ± 5.48) and vector group (134.40 ± 14.79) (t =15.51, P < 0.001, Fig. 3). The results showed that down regulating the expression of CHKA could signi cantly inhibit the clonal ability of glioma cells.

3.4Down regulation of CHKA expression can signi cantly inhibit the invasion of glioma cells
In order to verify the relationship between the expression of CHKA an the invasive activity of glioma cells, Transwell assay was performed on U251 glioma cell transfected with CHKA silencing lentivirus and its con-trol cell (shCHKA group and vector group). The number of invasive cells in CHKA group was (36.20 ± 11.82) and that of vector group was (104.40 ± 19.92), the difference was statistically signi cant (t = 6.58, P < 0.001, Fig. 4). The results showed that down regulating the expre-ssion of CHKA could signi cantly inhibit the invasion of glioma cells.

3.5MN58b, an inhibitor of CHKA, can inhibit the proliferation of glioma cells
We treated U251 glioma cells with different concentrations of MN58b (10 μ m, 20 μ m, 40 μ m) dissolved in DMSO solution, and then treated with CCK8 at different time points (24h, 48h, 72h, 96h).The absorbance of each group at 450 nm was detected (Fig. 5). The results showed that the proliferation ability of glioma cells was signi cantly inhibited with the increase of drug concentration and the prolongation of action time, which indicated that the proliferation of glioma cells could be inhibited by using MN58b, which is a CHKA inhibitor.

3.6MN58b, an inhibitor of CHKA can effectively inhibit the invasion of glioma cells
In order to verify the effect of MN58b, an inhibitor of CHKA, on the invasive activity of glioma cells, U251 cells were treated with 10μm MN58b and DMSO for 48 hours, then Transwell test was performed.
Transwell invasion assay showed that the number of invasive cells in shCHKA group was (56. 20 ± 9.42) and that of vector group was (188.60 ± 10.24), the difference was statistically signi cant (t = 21.28, P < 0.001, Fig. 6). The results showed that MN58b, an inhibitor of CHKA, could effectively inhibit the invasion of glioma cells.

MN58b, an inhibitor of CHKA can inhibit the EMT progression of glioma cells.
Through the tumor formation experiment in nude mice, we found that there were signi cant differences in the immunohistochemical results of EMT speci c molecules E-cadherin, N-cadherin and Vimentin between the experimental group treated with MN58b and the control group injected with normal saline. The results showed that the positive area of epithelial cell adhesion protein E-cadherin in the experimental group was 29.74 ±1.42 and that in the control group was 22.30±1.49, p < 0.05 .The difference was statistically signi cant (Fig. 7). The positive area of mesenchymal cell characteristic protein N-cadherin and Vimentin in the experimental group was 24.39 ±1.23 37.28 ±0.51 and that in the control group was 33.79 ±0.89 , 47.41 ±0.39 p < 0.01 .There was signi cant difference between the two groups (Fig.  7). The results showed that the expression of epithelial marker E-cadherin increased, the expression of mesenchymal markers N-cadherin and Vimentin decreased, the cell adhesion was strong, the invasive ability was weakened, and the process of EMT was inhibited in glioma cells.

Discussion
Multivoxel 1 H-MRS can detect the chemical components of brain cell metabolism noninvasively, and can evaluate and diagnose diseases according to their quantitative changes. It has become an important technical means for glioma research, clinical diagnosis and treatment [19,20]. In this study, we rst used multivoxel 1 H-MRS to identify the regions of Cho / Cr metabolism differences in the tumor cells and fuse the collected data with the neuro-navigation system. Neurosurgeons with many years of clinical experience accurately selected the tumor tissues with the highest and lowest Cho / Cr levels as clinical samples for real-time PCR and Western blot studies. The results showed that CHKA was found in Cho / Cr in glioma tissues with high metabolism was signi cantly higher than that in glioma tissues with low Cho / Cr metabolism. In recent years, a number of studies have found that Cho / Cr is related to the invasion and proliferation of glioma cells, and the increase of Cho / Cr is more common in high-grade gliomas [21][22][23]. Therefore, we infer that CHKA may be related to the multivoxel 1 H-MRS parameter Cho / Cr, and affect the occurrence and development of glioma through the role of Cho metabolism. On the one hand, we used lentiviral transfection to down regulate the expression of CHKA in U251 glioma cells, and on the other hand, we treated U251 glioma cells with MN58b, a speci c inhibitor of CHKA, to reduce the biological activity of CHKA. Furthermore, cloning experiments, CCK8 and Transwell experiments proved that the ability of U251 cells to clone, proliferate and invade after the down-regulation of CHKA gene and the inhibition of biological activity of CHKA were con rmed to be inhibited.
As an important enzyme in Cho metabolism, CHKA has been widely concerned about its role in human tumorigenesis and development [24]. Recent studies by some sCholars have shown that the expression level of CHKA in a variety of tumor tissues is signi cantly increased by [25,26], and our experimental results are consistent with it. It has also been shown that CHKA interacts with a variety of tumor related signaling pathways, which can be activated by a variety of proto oncogenes. In breast cancer [27], CHKA promotes the proliferation of breast cancer cells through c-Src dependent protein interaction with EGFR. In cervical cancer [28], as a catalytic enzyme, indirectly affects Akt, MEK / ERK signaling pathways by regulating the production of phosphatidylCholine, Cytidine Diphosphate Choline in Choline metabolic pathway, and ultimately promotes the occurrence and development of cervical cancer. In T-cell lymphoma [29], CHKA is involved in the regulation of ras-akt / ERK signaling pathway, thus affecting the apoptosis and necrosis of T-lymphoma cells. In order to explore its role and mechanism in glioma, we searched the literature related to glioma and found that Katharina Koch et al reported in 2016 that CHKA is an important regulator of EMT in glioblastoma [30], but how it regulates and its speci c mechanism has not been systematically studied.
In recent years, a large number of literature reported that there are a small number of stem cell characteristic cells in a variety of tumor cells [31,32]. These cells have the potential of self-renewal and multi-directional differentiation, which are de ned as cancer stem cells [33]. The origin of these tumor stem cell subpopulations is still unclear. It may originate from mutated normal stem cells, dedifferentiated somatic cells or cancer cells undergoing epithelial mesenchymal transition. Studies have shown that there are stem cell like tumor initiation cells in glioblastoma, called glioma stem cell [34]. Glioblastoma stem cells can self-renew and proliferate stably. They can form neurospheres in speci c growth environment and have strong tumorigenicity [35]. Studies have found that compared with normal glioma cells, glioma stem cells are more resistant to radiotherapy and chemotherapy. Glioma stem cells may be the source of the occurrence and development of glioma, and are closely related to the poor prognosis of glioma [36]. Therefore, the research of glioma stem cells has become the current trend and focus of malignant glioma treatment.
In our previous studies, we found that the regions of Cho / Cr metabolism difference of multivoxel 1 H-MRS parameters were closely related to the enrichment of tumor stem cells in glioma [37]. The preliminary study con rmed that there were a small amount of glioma stem cells in the brain glioma tissue of Cho / Cr low metabolism area, while the content of glioma stem cells in Cho / Cr high metabolism area was signi cantly higher than that in Cho / Cr low metabolism area. The distribution of glioma stem cells was positively correlated with Cho content. This is consistent with the expression of CHKA in the region of Cho / Cr metabolism difference of multivoxel 1 H-MRS parameters. Therefore, we speculate that there may be some relationship between CHKA and glioma stem cells, Katharina Koch. Our results suggest that CHKA is an important regulator of EMT in glioblastocytes, and EMT, as a key transformation pathway for malignant tumor cells to acquire invasiveness, may be an important reason for therapeutic resistance and tumor recurrence of glioma stem cells [38].Our experimental results also show that CHKA is related to glioma stem cells and its inhibitor MN58b can slow down the progress of EMT,these are consistent with our conjecture.

Conclusion
In , as a tumor promoting gene, CHKA plays an important role in the proliferation, cloning, invasion and other processes of glioma. We speculate that CHKA may be a pleiotropic negative regulatory factor related to glioma growth, which can induce EMT of glioma cells, thus endowing the transformed glioma cells with stem cell like characteristics. Gliomas with similar characteristics can lead to the occurrence and progression of tumors. The expression of these glioma stem cells and CHKA in glioma tissues can be detected non-invasively by multivoxel 1 H-MRS parameter Cho / Cr, which introduces a new theory for the treatment, is expected to guide the surgical resection range, improve the therapeutic effect and thereby, help improve the overall prognosis of Gliomas.