LncRNA CASC2 Inhibits Progression of Glioblastoma by Regulating the Expression of AKT in T98G Cell Line, Treated by TMZ and Thiosemicarbazone Complex

Background: The aim of this study was to evaluate the expression alterations of CACS2 and its target gene, AKT, in T98G cell line treated with Temozolomide and Thiosemicarbazone complex (Ni, Cu) and to compare the results with each other. Methods: Temozolomide and Thiosemicarbazone complexes were prepared in different concentrations. Cell culturing of T98G cell line was carried out and was classified into 3 groups based on the incubation time (24, 48, and 72h) with utilized agents, after RNA extraction the expression level of CACS2 and AKT genes were evaluated by Real-time PCR. Ultimately, the results were analyzed by Rest software. Results: CASC2 expression under Temozolomide treatment at different concentrations (100, 150, 200, and 250 µM) and different time periods (24, 48, and 72h) was increased. Moreover, its expression was significantly upregulated after treating with Ni at the concentrations of 100.5 and 104 µM after 24h. Furthermore, its expression was augmented after 72 h Cu treatment at the concentrations of 15, 16, 17, and 18 µM. In addition, AKT expression after Temozolomide and Thiosemicarbazone complex treatment was significantly decreased (P <0.001). The results showed that the expression alterations of CASC2 and its target gene, AKT, after treatment with Temozolomide and Thiosemicarbazone are highly depended on incubation time and concentration. Conclusion: In a conclusion, the studied agents at different concentrations and times showed a high potential to control the expression of the studied lncRNA and gene in glioblastoma cells.

As for the first time, cancer susceptibility candidate 2 (CASC2) lncRNA was known as a tumor suppressor non-coding RNA in human endometrial cancer (Yu et al., 2018). CASC2 generates three alternative transcripts: CASC2a, CASC2b, and CASC2c, and in fact, it is the lncRNA CASC2a that functions as a tumor suppressor in malignancies like glioma (Palmieri et al., 2017) (Palmieri et al., 2017). lncRNA CASC2, on the other hand, is elevated in glioma and promotes carcinogenesis. CASC2a and CASC2c are both derived from CASC2, but only share the first three exons and have different downstream exons. CASC2a inhibits glioma development, whereas CASC2c functions as an onco-RNA (Liu et al., 2017) . It has been demonstrated that the mediated prohibition of glioma growth, migration, invasion, and cell apoptosis by CASC2a is linked to miR-21 dysregulation. By functioning like a miR-101 decoy sponge, CASC2c promotes astrocytomic carcinogenesis (Junyang et al., 2018). Moreover, by inhibiting miR-181a expression, lncRNA CASC2 can inhibit the AKT/PI3k signaling pathway. AKT activation is one of the most frequent molecular cancer changes (Bellacosa et al., 2005). The AKT pathway is thought to be a significant cancer therapy target. Overexpression of CASC2 can also inhibit cancer development by suppressing EIF4A3 expression and blocking the PI3K/ AKT/mammalian target of rapamycin (mTOR) pathway .
One chemotherapy medication for treatment of glioblastoma is an alkylating agent, termed Temozolomide (TMZ), which can convert imidazole 4 Carboxamide into active 5-(3-methyltriazen 1-yl) metabolite, causing cell cycle arrests and eventually cell death by destroying double DNA strands (Lee, 2017). TMZ transforms into the active metabolite 5-(3-methyltriazen-1-yl) imidazole-4carboxamide by non-enzymatic chemical transformation, which reason cell cycle arrestment and finally cell death by breaking the DNA double strands (Lee , 2017;Thomas et al.,2017). Even in older glioblastoma patients, the TMZ can be tolerated (Behm et al.,2013). TMZ, which is an alkylating agent with the ability in causing DNA damage in the malignant cells, is increasing median overall survival from 12.1 to 14.6 months in normal postoperative radiation therapy (60 Gy.30 fractions) (Stupp et al., 2005). Thiosemicarbazone's anticancer effects were merely once ascribed to the inhibition of ribonucleotide reductase, a rate-limiting enzyme engaged in the synthesis of DNA. Thiosemicarbazone's capacity to chelate metal ions is now recognized as the primary factor in its anti-proliferative actions. Thiosemicarbazone complexes' redox activity is crucial to their anticancer function, causing oxidative damage and blocking ribonucleotide reductase's activity. In vivo studies show that many Thiosemicarbazones have the potential to be used as chemotherapeutic agents (Kalinowski et al., 2009). The coordination of metal ions such as Cu, Ni, Zn, and Pd can improve the cytotoxic actions of these ligands, which can also increase their lipophilicity and mechanism of action within the cell (Jayakumar et al., 2014).
The primary goal of this study was to examine at the altered expression of the AKT gene and CASC2 lncRNA after they were treated with the chemotherapeutic drug TMZ and the Ni, Cu complex of Thiosemicarbazones. Furthermore, we investigated the therapeutic efficiency of utilized agents in different incubation times and concentrations. In addition, the effect of TMZ and Thiosemicarbazones complexes of Ni and Cu on changing the expression levels of AKT gene and CASC2 lncRNA was compared. The relationship between expression alterations of AKT and CASC2 with each other (whether the increased expression level of CASC2 at the same time and concentrations can terminate to decreased expression level of AKT or not) was examined. Last but not least, the optimal time and effective dosage in which utilized agents could alter the expression level of AKT and CASC2 was determined.

Materials and Methods
The primary goal of this study was to examine at the altered expression of the AKT gene and CASC2 lncRNA after they were treated with the chemotherapeutic drug TMZ and the Ni, Cu complex of Thiosemicarbazones. This study conducted from April to September 2019 in Zanjan Islamic Azad University Research Center for Science. Human glioblastoma cell line T98G was prepared from the Pasteur Institute of Iran in the first cellular passage and at a density of approximately 80% of cells (2×10 5 cell.cm 2 ).

T98G Glioblastoma Cell line and cell culture
Cell culturing, passaging, cell counting and preparation of different concentrations of the abovementioned drugs were performed. Human Glioblastoma cell line, T98G was first cultured in Dulbecco's Modified Eagle's Medium (RPMI 90%) medium containing 10% FBS, followed by incubation at 37 °C containing 5% carbon dioxide for six days. Subsequently, three passages were applied to the cells at two consecutive intervals and at each passage period, the resulting cells were transmitted into the flask with fresh culture medium. Finally, the fourth cell passages were selected for next investigations. The aggregated cells were counted by trypan blue staining method. After cell counting, the number of cells were 3×10 4 cells per cm 2 . Subsequently, the cells were divided into two groups, including the control and under treatment groups.

TMZ and Thiosemicarbazone complex (Ni, Cu) preparation and drug treatment
In order to prepare the TMZ drug, first 9cc sterile distilled water with 1.5cc DMSO were mixed and 105 µM of this mixture was obtained and 1 tablet of TMZ was dissolved in it. To construct Ni stock, 000.1 g of Ni was dissolved in 1 cc of water. For preparing Cu exactly the same amount of utilized Ni was dissolved in 1 cc of distilled water, and then effective doses of the drugs (TMZ and Thiosemicarbazone complex Ni, Cu) were examined by MTT test.
The detailed information about their concentration and incubation time for prepared drugs is listed in Table 1.

RNA extraction and cDNA synthesis
Following cell treatment according to the groups were inserted and amplified in real-time PCR. The primer sequences used for the CACS2 lncRNA, AKT and GAPDH target gene is listed in Table 2.

Gene Expression Confirmation and Statistical Analysis
To confirm the presence of the studied genes, PCR products were electrophoresed on 2% agarose gel and the length of the fragments were equal to 125 bp, sequenced and confirmed by Fan Avaran Gene Company (Ebrahimi et al., 2022). After reaction, the difference of target gene CT to the reference gene was calculated as Δct for each sample. Then, 2ΔΔct (fold change) was obtained for each sample. To investigate the expression of CACS2 lncRNA and AKT target gene by Real Time PCR, LIVAK method (Livak method) and Rest (2002) were used. P <0.05 was considered as the accepted cutoff criteria of significance rate.

Results
Expression changes of lncRNA CASC2 treated with TMZ and Thiosemicarbazone complexes (Cu, Ni) CASC2 expression in Glioblastoma cells after 24 h treatment with TMZ at different concentrations of 100, 150, 200, and 250µM, showed increased expression level with the rates of (2.41), (2.75), (2.268) and (5.934) respectively. This gene's expression level was also increased after 48h treatment and at the abovementioned concentrations showed (1.304), (1.899), (1.219) and (2.406) expression change level rates. 72 h treatment of the cell line through the same concentrations also showed increased change levels by the rates of (4.075), (1.264), (2.383), and (3.373) respectively (P<0.001). (Figure 1) The expression changes levels of CASC2 treated with Thiosemicarbazone is summarized at Table 3 and Figure  2, 3.
Expression changes of AKT treated with TMZ and Thiosemicarbazone complex (Cu, Ni) AKT gene expression was significantly reduced in listed above, RNA extraction and cDNA synthesis were carried out. After treating the cells with the afore-stated time points (24,48,72), in order to evaluate expression of the studied genes after and before treatment, the RNA Extraction (by RNA -Roche-High Pure Nucleic Acid Kit (Gmbh, Manheim, Germany, cat.no 11858874001)) and cDNA synthesis process (Takara Bio, Otsu, Japan, cat.no RR037Q) were performed according to the kit instructions.

Quantitative real-Time PCR (Q-PCR) and RNA expression profiling
The below-mentioned steps and compounds are required for Real-Time PCR process which were prepared for CACS2 lncRNA and the AKT target gene: The FASTA sequence of the cDNA library of genes were extracted from the NCBI website. Primer3 software was used for primer designing procedure. Finally, using the software mentioned above, the results were blasted on the NCBI website to confirm specificity and primer binding to all three studied genes. The reactions were adjusted at 25 µl volume. 12.5µl of Cyber Green (1X), 1 µl of forward and reverse primers (0.4µM), 2µl of cDNA, and 8.5µl of deionized water were mixed. The first denaturation schedule was at 95 °C for 10 min and DNA fragments were amplified in 40 cycles (denaturation of 95 °C for five seconds, 51.8 annealing, 30 seconds for the AKT, and, lncRNA CASC2, and final extension 72 °C, 30 seconds.). Using Rotor-Gene Q real-time PCR cycler (Qiagen, Hilden, Germany) and (Takara Bio, Otsu, Japan, Cat # RR820Q) Sybergreen kit instruction, CACS2 lncRNA and AKT and GAPDH target genes as reference genes   (Figures 1,2,3).

Discussion
Gliomas constitute the most common primary brain tumors and the most lethal central nervous system cancers (Chen et al., 2013;Shen et al., 2019). Many studies have found a relationship between lncRNAs and glioma. A research, , discovered that a variety of lncRNAs are implicated in the beginning of glioma (Li et al., 2019). The goal of this work was to evaluate the expression alteration of lncRNA CASC2 in glioma cell line impacted by TMZ and Thiosemicarbazone Complexes.
In the present study, we showed that the alteration in the expression of lncRNA CASC2 and AKT after TMZ and Thiosemicarbazone complex of Cu/Ni treatment differs with each other. We also demonstrated that the Thiosemicarbazone complex Cu had the largest effect on boosting lncRNA CASC2 expression in contrast to TMZ and performed better and less effectively than TMZ. In addition, Ni, when compared to TMZ, had an impact at lower concentrations and for a shorter period of time. The comparison of the effects of TMZ, Ni, and Cu on AKT expression revealed that the Thiosemicarbazones complex Cu had the highest effect on increasing AKT expression in comparison with TMZ and performed better at lower concentrations and faster time than the TMZ medication.   Ni also needed lower concentrations and a shorter half-life than TMZ. As a result, it can be concluded that the Ni and Cu were more potent than TMZ in changing AKT expression.
CASC2 and mir21 lncRNAs have been found to have an inhibitory impact on each other's expression in studies. CASC2 promotes gene transcription and cell survival by decreasing mir21 expression via the mir21 / PTEN / AKT / mTOR axis and the NF-κB signaling pathway (Feng et al., 2017). Furthermore, CASC2 inhibits the mir-18 / PIAS3 / STAT3 axis, which promotes cell survival (Huang et al., 2016). CASC2, on the other hand, promotes cell death by suppressing mir21 expression via the pdcd4/p21 axis. Controlling variations in CASC2 expression and its   CASC2 expression was shown to be significantly lower in cisplatin-resistant NSCLC tissues and cell lines, according to XIAO and colleagues in 2020. MiR-18a, which was upregulated as CASC2 expression decreased, lowered the protein content of IRF-2 in NSCLC cells. Furthermore, the transcription factor ELF1 was discovered to be a CASC2 promoter and to enhance its expression levels in cisplatin-resistant NSCLC cells (S-YH, 2020). Zhu & al. reported in 2019 that CASC2 over-expression improved the cisplatin's inhibitory impact on cell viability and increased the release and apoptosis of cisplatin-induced LDH. They discovered that CASC2 inhibited the AKT pathway via suppressing miR-181a expression (Dengyan et al ., 2019 ) . In 2018, Jin and coworkers discovered a CASC2/miR-24/miR-221 axis that influences TRAIL resistance in hepatocellular carcinoma (Xiaoxin et al., 2018). Wang and colleagues stated in 2017 that they were researching CASC2 RNA as a glioma suppressor in the Wnt/-catenin signaling pathway. CASC2 overexpression significantly reduced glioma cell proliferation, migration, and invasion by inhibiting the Wnt/-catenin signaling pathway (Ronglin et al., 2017) . Their findings suggest that activating the AKT/mTOR pathway inhibits cisplatin-induced apoptosis, resulting in cisplatin resistance. As a result, their findings show that cisplatin resistance in human ovarian cancer cells can be reversed by targeting the AKT/mTOR survival pathway (Peng et al., 2010). In 2005, Li and colleagues published a study and demonstrated that MCF7, MDA468 and T47D cells show a drug-dose-dependent rise in phosphorylated AKT levels within 24 hours after doxorubicin treatment. Phosphorylation of AKT by doxorubicin was linked to enhanced kinase activity and was reliant on phosphoinositide 3-kinase (PI3-K) (Xinqun et al., 2005) .
The results of a study on the changes in the expression of the CASC2 tumor suppressor lncRNA and the AKT oncogene treated with the mentioned drugs, as well as a comparison of the findings with previous studies, revealed the importance of the AKT oncogene in the PI3K/AKT signaling pathway and changes in the expression of the CASC2 tumor suppressor lncRNA in proliferation and apoptosis of all cancer types. The findings highlight the significance of increasing in the expression of the AKT oncogene and decreasing the expression of the CASC2 tumor suppressor gene in glioblastoma patients treated with Thiosemicarbazones complex Ni and Cu. We discovered that these drug compounds had a positive effect on changes in the expression of these genes.
Finally, lncRNA CASC2 is one of the key role players involved in tumor suppression in glioblastoma. In terms of effectiveness and influence on changes in the expression of the investigated genes, evidence revealed that the Thiosemicarbazones complex Ni and Cu, as well as the standard medication TMZ, depended on time and concentration. Increases in the duration and concentration of the cell line's treatment with the indicated medicines, on the other hand, resulted in significant alterations in the expression of the AKT gene. In compared to the control groups, the outcomes of the medication combinations exhibited two types of rising and lowering impacts. CASC2 tumor suppressor lncRNA and AKT oncogenes expression changes had a beneficial influence on regulating the expression of the genes examined. These alterations suggested that the investigated pharmacological compounds had a non-random and focused influence on gene expression changes. These effects might vary depending on the concentration and duration. Therefore, the studied drugs, including Thiosemicarbazone complex Cu and Ni, had good potential to control changes in the expression of the studied genes, which confirms the effectiveness of these compounds in controlling changes in gene expression due to mutations, treatment, and reduction of glioblastoma progression associated with changes in the expression of suppressor and oncogene genes including CASC2 and AKT.

Author Contribution Statement
Study concept and design: G. A.T; Acquisition of data: Sh. Sh; Analysis and interpretation of data: G. A.T; Drafting of the manuscript: N. Z; Critical revision of the manuscript for important intellectual content: S.M, G. A.T; Administrative, technical, and material support: Sh. Sh.