Bioinformatics analysis

In our research, the data employed was sourced from two publicly accessible databases: the Chinese Genome Glioma Atlas (CGGA) and the Cancer Genome Atlas (TCGA). The TCGA database offered RNA - sequencing data along with clinical information from 702 patients. Conversely, the CGGA database furnished RNA - sequencing data and clinical particulars from 325 patients. Our research project adhered to the Helsinki Declaration and received authorization from the ethics review panel of the Chinese PLA General Hospital. This authorization confirmed that the patients’ informed consent had been secured for both public data repositories. Subsequently, we examined the associations between the expression amounts of SIRT7 messenger ribonucleic acid (mRNA) and typical prognostic characteristics. such as the World Health Organization classification, IDH wildtype/mutant, 1p/19q coding/non-coding, MGMT methylated/un-methylated. In addition, we analyzed the relationship between patients’ clinical prognosis and SIRT7 expression.

Tissue samples and cell culture

A total of 18 samples of primary glioma tissue and 6 samples of non - neoplastic brain tissue were procured by the neurosurgery department at the First Medical Center of the Chinese PLA General Hospital. Normal human astrocytes (NHA) and several human glioblastoma cell lines were furnished by the cell bank of the Chinese Academy of Sciences in Shanghai, China. The cell lines consisted of U87MG, U251, A172, U118MG, T98G, and LN229. Cell lines of glioblastoma were grown in Dulbecco’s Modified Eagle’s Medium that had a high level of glucose (Product No. 21013024, Gibco, USA). To this culture medium, 10% Fetal Bovine Serum (Product No. A5670701, Gibco, USA) and 1% penicillin/streptomycin (Product No. 15140122, Gibco, USA) were added. Conversely, NHA cells were incubated in astrocyte medium (Product No. #1801, ScienCell, USA). Each individual cell was maintained at a temperature of 37 degrees Celsius within a humidified incubator that had an atmosphere consisting of 5% carbon dioxide.

Reagents and transfection

Specifically, we explored whether microRNAs may post-transcriptionally regulate SIRT7 expression, TargetScan 7.2, starBase v3.0 and miRDB were utilized to predict target mircoRNAs. The miR-148a-3p mimics, miR-148a-3p inhibitors, microRNA negative control (miR-NC), and inhibitor negative control (inhibitor-NC) were procured from OBiO Technology, a company located in Shanghai, China. Genechem, another Shanghai-based firm in China, supplied the lentivirus packages of SIRT7 short hairpin ribonucleic acid (shSIRT7), SIRT7 negative control (shNC), along with the pcDNA - SIRT7 overexpression plasmid (OE) and its corresponding negative control (OENC). Temozolomide (TMZ) was acquired from Selleck.cn (product number S1237, China). In accordance with the instructions furnished by the manufacturer, the glioblastoma cells underwent transfection using the Lipofectamine™ 3000 transfection reagent (product number L3000015, Thermo Fisher Scientific, USA). These cells were transfected with either 100 nanomoles per liter (nM) of miR-148a-3p mimics or 200 nM of miR-148a-3p inhibitors. A ratio was set where for every 0.04 nanomoles (nmol) of miRNA mimics or inhibitors, 2.5 µl (µl) of the transfection reagent was used.

Reverse transcription-quantitative PCR (RT-qPCR) assay

According to the manufacturer’s guidelines, the TRIzol™ Reagent (15596026CN, Invitrogen, USA) was used to extract ribonucleic acid (RNA) from clinical tissue specimens and cells cultured in the laboratory. To quantify the expression level of miR-148a-3p, the TaqMan MicroRNA Assay kit (4427975, Thermo Fisher Scientific, USA) was employed. The All - in - One™ miRNA First - Strand cDNA Synthesis Kit 2.0 (Product Code QP113, produced by GeneCopoeia in China) was utilized to transform the extracted ribonucleic acid (RNA) into complementary deoxyribonucleic acid (cDNA) via reverse transcription. Subsequently, the PowerTrack™ SYBR Green Master Mix, which is intended for quantitative polymerase chain reaction (qPCR) (Product Code A46109, provided by Thermo Fisher Scientific in the USA), was applied for quantification on the Roche LightCycler480 system (Product Code 900066, made by Roche in Switzerland). Glyceraldehyde 3-phosphate dehydrogenase(GAPDH), functioning as a constitutive gene, was employed to standardize the expression of Sirtuin 7(SIRT7).The sequences of the quantitative primers for SIRT7 are presented below.The forward primer had the sequence 5’-AGCACGGCAGCGTCTATC-3’,and the reverse primer had the sequence 5’-CATGTGGGTGAGGGTTGG-3’.Regarding GAPDH, the forward primer had the sequence 5’-TCCACATGAAGTGTGACGT-3’,and the reverse primer had the sequence 5’-TACTCCTGCTTGCTGATCCAC-3’.

Western blot assay

A mixture of RIPA extraction buffer (R0278, Sigma - Aldrich, Germany) and a protease inhibitor cocktail (P1011, Beyotime, China) was employed to lyse cells and tissues. Subsequently, the BCA protein assay kit (PC0020, Solarbio, China) was utilized to measure the protein concentrations of the test samples. A quantity of 30 micrograms of protein was subjected to separation via sodium dodecyl sulfate - polyacrylamide gel electrophoresis. Subsequently, the separated proteins were transferred onto PVDF membranes (88518, Thermo Fisher Scientific, USA). To begin with, the PVDF membranes were subjected to a blocking procedure using 5% Bovine Serum Albumin (BSA) (product number ST023, Beyotime, China). Following this, these membranes were incubated at 4 degrees Celsius overnight with primary antibodies. The specific primary antibodies employed in this experiment were those targeting SIRT7 (product number 5360, CST, USA) and GAPDH (product number 2118, CST, USA). Afterward, a secondary antibody, namely a goat - anti - rabbit antibody conjugated with horseradish peroxidase (HRP), was added to bind to the primary antibodies (7074 and 7076, CST, USA). Next, the PVDF membranes were permitted to react with Immobilon Western Chemiluminescent HRP Substrate (P90719, Millipore, USA) for a period ranging from 10 to 15 s. Ultimately, a quantitative densitometry analysis of the protein bands was performed using Quantity One Software (Bio - rad, USA).

Luciferase reporter assay

The psiCHECK − 2 luciferase vector was engineered to incorporate segments of the wild - type SIRT7 3′ untranslated region (UTR). It is hypothesized that these segments harbor binding sites for miR-148a-3p. Additionally, the vector was built to include their mutated counterparts (Promega, Madison, WI, USA). After that, luciferase reporters for SIRT7 3’ UTR - wild type (WT), SIRT7 3’ UTR - mutant 1 (MUT1), and SIRT7 3’ UTR - mutant 2 (MUT2) were produced respectively. Next, glioblastoma cells that had been transfected with miR-148a-3p mimics and the negative control miR - negative control (NC) were co - transfected with the previously mentioned reporters. Forty - eight hours after transfection, a dual luciferase reporter assay system from Promega was utilized to measure the luciferase activities.

Cell counting kit-8 (CCK-8) assay

The capacity of glioblastoma cells to multiply was determined using the CCK − 8 assay (C0041, Beyotime, China). Specifically, the cells were transferred into 96 - well plates. After the cells had been infected or treated, 10 µl of CCK − 8 solution was introduced into each well. Subsequently, the plates were incubated at 37 °C for a duration of 2 h. Following this, the absorbance at a wavelength of 450 nm (OD450) was measured.

5-ethynyl-2’-deoxyuridine (EdU) assay

The BeyoClick™ EdU − 594 cell proliferation assay kit (C0078L, Beyotime, China) was employed to measure the DNA synthesis of glioblastoma cells. One day before the experiment, glioblastoma cells were plated into 24 - well plates. Then, following the instructions provided by the product’s manufacturer, the pre - heated EdU working solution was added. After adding the solution, the cells were placed in an incubator maintained at 37 °C for 2 h to perform EdU labeling. Once the labeling was completed, the cells were fixed with a 4% paraformaldehyde solution. Subsequently, 500 µl of the Click reaction solution was added, and the cells were incubated in the dark at room temperature for 30 min. After that, 1 milliliter of the Hoechst 33,342 solution was added, and the incubation was continued in the dark at room temperature for 10 min. Eventually, the cells were examined using a fluorescence microscope (Olympus BX51, Japan).

Mouse experiments

All experimental procedures were conducted in strict accordance with the Guidelines for the Welfare and Utilization of Laboratory Animals promulgated by the National Institutes of Health. Moreover, these procedures received authorization from the Animal Experiment Ethics Committee of the Chinese PLA General Hospital (Authorization number: 2018–066). Four groups were randomly formed using 3–5 - week - old male Balb/c nude mice. The first cohort was the control cohort, simply named “Control”. The second was the temozolomide (TMZ) cohort. The third was the shSIRT7 cohort, and the fourth was the TMZ + shSIRT7 cohort. Each of these cohorts was composed of three mice. Subsequently, 5 million U251 cells were separately injected beneath the skin of the left upper limb of each mouse. One week after the injection of cells, temozolomide (TMZ) was administered via intraperitoneal injection. The dose was established at 20 milligrams per kilogram of the body’s weight, and this therapy was conducted on a daily basis for a span of two weeks. Thirty - five days after the initial cell injection, the tumors were excised surgically and then measured for weight.

Immunohistochemistry

The tissue specimens were immobilized with a 4% paraformaldehyde solution. Subsequently, they were encased in paraffin, and slices of suitable thickness were obtained using a microtome. At room temperature, the slices were deparaffinized using xylene and then rehydrated with absolute ethanol. Antigen retrieval was achieved by immersing the slices in a sodium citrate solution within a 98℃ hot water bath for 15 min. To avoid non - specific attachment, a goat serum blocking solution was applied at ambient conditions. The tissue specimens were exposed to a SIRT7 antibody (product code 12994–1 - AP, sourced from Proteintech, USA) and a Ki − 67 antibody (product code 27309–1 - AP, also procured from Proteintech, USA). The specimens were then subjected to an overnight incubation at 4℃ to enable the antibodies to interact with the sections. On the subsequent day, the slices were initially stored at ambient temperature for half an hour. Subsequently, the antibodies were eliminated via a washing process. Afterward, 50 µl of a Streptomyces antibiotic - peroxidase mixture was added and incubated at room temperature for 10 min. Ultimately, this mixture was washed away. Two droplets of DAB solution, totaling 100 µl, were added to each slide. After that, the slides were put under a microscope for a 10 - minute examination. Once the staining reached an ideal state, the staining process was halted, and the slides were washed. Hematoxylin was used for counter - staining, and the counter - staining lasted for 2 min. As soon as the cell nuclei turned blue, the counter - staining was ended, and the slides were washed once more. Finally, the slices were left to air - dry before being photographed.

Statistical analysis

Each experiment was carried out independently a minimum of three times. All the data mentioned earlier was analyzed using GraphPad Prism8 software. To assess the differences statistically, the Student’s t - test, one - way ANOVA, and the Tukey post - hoc test were employed. A P - value less than 0.05 was regarded as statistically significant.