W15 Krško stats & predictions

Il Programma delle Partite

• Mattinata: I match iniziano nelle prime ore della mattinata con partite destinate a definire gli equilibri del torneo. Le partenze sono decise e i giocatori sono motivati a lasciare il segno sin dall'inizio.
• Pomeriggio: Il pomeriggio promette incontri combattutissimi, con tanti top seed che si confrontano sotto il sole caldo della Slovenia. Questo è il momento perfetto per i fan che vogliono vedere le vere sfide tra i migliori talenti.
• Sera: I match serali offrono una piacevole conclusione alla giornata sportiva, con partite che possono decidere le sorti di alcune qualificazioni cruciali. L'atmosfera è elettrizzante e gli spettatori godono della vista delle luci che illuminano il campo, creando un contesto scenografico mozzafiato.

Analisi Dettagliata dei Match di Oggi

Entriamo nel dettaglio delle partite previste per la giornata di domani, identificando le chiavi di accesso e offrendo previsioni puntuali per gli appassionati delle scommesse sportive.

• Giocatore A vs Giocatore B: La partita si annuncia come una battaglia tra tradizione e nuovi talenti. Il Giocatore A, un veterano del torneo, si trova di fronte a un giovane promettente che ha fatto parlare di sé nei tornei minori. La previsione è favorevole al Giocatore A, grazie alla sua esperienza, ma il calore della casa potrebbe certamente galvanizzare il giovane.
• Giocatrice C vs Giocatrice D: Entrambe le giocatrici vantano un ottimo record in superfici veloci. L'analisi tecnica suggerisce che la Giocatrice C possa avere un leggero vantaggio grazie alla sua miglior performance su cemento. Tuttavia, la Giocatrice D non soffrirà per questa differenza e potrebbe sorprendere con colpi vincenti.
• Giocatore E vs Giocatore F: L'incontro più atteso dell'intero programma. Il Giocatore E, favorito dai bookmaker per la sua forma fisica ottimale, viene sfidato da un Giocatore F con un animo da fuori di casa. La chiave del match è la resistenza, e chi riuscirà a mantenere il ritmo più alto avrà buone possibilità di prevalere.

Strategie di Scommessa Consigliate

Per chi è interessato a combinazioni vantaggiose nelle scommesse sportive, ecco alcuni consigli per massimizzare i profitti dalla giornata di domani.

• Over/Under Segna: Per il match tra Giocatore A e Giocatore B, si consiglia l'under, data l'alta probabilità di partita equilibrata e potenziali tie-break.
• Win-to-Loss Ratio: Seguire la strategia di puntare sul 3-1 per il match tra Giocatrice C e Giocatrice D potrebbe risultare fruttuoso, considerando l'equilibrio nel loro confronto recente.
• Prop Bets: Un'opzione interessante è puntare sul set vinto per primo, visto il 1: # Invasion of colorectal cancer cells mediated by exosomal miR-1307-3p via the downregulation of AKT3 2: Author: Zi-yi Zhao, Xue-feng Zou, Ai-xin Chen, et al. 3: Date: 12-24-2022 4: Link: https://doi.org/10.1186/s12876-022-02577-y 5: BMC Gastroenterology: Research 6: ## Abstract 7: BackgroundColorectal cancer (CRC) is a common gastrointestinal malignancy with high malignancy and recurrence rates. Multiple studies have demonstrated that miR-1307-3p plays an important role in the progression of various types of cancer. However, the precise role of miR-1307-3p in CRC remains to be determined. 8: PurposeTo investigate the role of miR-1307-3p on CRC progression through recombinant lentivirus transfection and exosomes isolation. 9: MethodsThe expression levels of miR-1307-3p and AKT3 between tumor tissue and adjacent non-tumor tissue were detected by RT-qPCR and Western blot assay. The effect of miR-1307-3p on proliferation and invasion of CRC cells was evaluated using Cell Counting Kit-8 (CCK8) and Transwell assays, respectively. Subsequently, the targeting relationship between miR-1307-3p and AKT3 was confirmed using the luciferase reporter assay. 10: ResultsWe observed that miR-1307-3p was elevated in CRC tissue samples and cell lines while an expression level in contrast tendency was displayed by AKT3. MiR-1307-3p promoted proliferation and invasion of CRC cells by targeting AKT3. Moreover, we identified that miR-1307-3p was elevated in CRC cell-derived exosomes while exosomal miR-1307-3p carried by macrophages promoted invasion of CRC cells. 11: ConclusionsOur data indicate that exosomal miR-1307-3p might promote the invasion of CRC cancer cells through downregulating AKT3 expression. 12: ## Introduction 13: Colorectal cancer (CRC) is a common gastrointestinal malignancy which poses a great threat to human health worldwide [1]. According to the latest data from Cancer Research UK (2021), there were more than 42 thousand new cases of CRC in 2020 in the United Kingdom and nearly over 16 thousand patients died from CRC in the same year [2]. Although vigorous efforts have been made for decades to develop effective strategies to prevent and treat CRC, the prognosis of CRC patients remains poor, especially those at late stages [3]. Previous studies have demonstrated that the 5-year survival rate for CRC patients in the early stage is up to 90%, while that for those in the late stage is less than 14% [4]. The overall development of CRC is a complicated process involving genetic mutations and epigenetic modifications [5]. In addition to genetic factors, studies have shown that epigenetically regulated microRNA (miRNAs) are also important players in CRC progression [6]. Therefore, it is necessary to further explore the epigenetic mechanisms and molecules involved in CRC progression. 14: miRNAs are small non-coding RNAs containing 18–25 nucleotides in length which can regulate gene expression by targeting the 3′ untranslated region (UTR) of specific mRNAs [7]. Multiple studies have demonstrated that miRNAs are important players in various physiological processes, especially they play vital roles in cancer initiation and progression [8, 9]. For instance, aberrant expression of miR-138 was related to a larger tumor size and higher ability of migration and invasion in lung cancer [10]. Dysregulation of miR26a-b impaired cell proliferation and invasion in liver cancer [11]. miR211 acts as a tumor suppressor by targeting HIF1α in hepatocellular carcinoma [12]. Moreover, increasing evidence suggests that deregulated expression of miRNAs is also involved in CRC initiation and development [13]. For instance, miR365 promotes colorectal cancer cell growth through the regulating HK2/PI3K/AKT pathway [14]. Upregulation of miR-139-5P repressed progression of colorectal carcinoma through targeting ZFAS1 [15]. MiR-374a inhibits colorectal cancer cell proliferation by targeting JAG1 [16]. 15: Recently, exosomes have received more and more attention due to their important role in cell-to-cell communication [17]. Exosomes are comparable small vesicles with a diameter range from 40 to 100 nm which are secreted by most kinds of cells [18]. In addition to the well-known function of exosome as vehicles for intercellular communication, it can also export cytosolic contents including proteins and various nucleic acid species into recipient cells [19]. Many studies have shown that exosomes can effectively mediate the transfer of tumor-related contents such as proteins and RNA between cells in various cancers [20, 21]. For example, exosomal miR1229 accelerates lung cancer progression by targeting PTEN [22]. Exosomal miR-125b released from preoperative biopsy promotes biliary tract cancer progression [23]. Recent studies have demonstrated that exosomal miRNAs play important roles in the regulation of CRC initiation and progression [24]. For instance, exosomal miR-197-3p is able to promote colon cancer development by targeting SMAD4 [25]. Exosomal miRNA-210 releases from CRC stem-like cells to promote invasion and metastasis of surrounding colorectal epithelial cells [26]. 16: Previous studies have revealed that miR-1307 is a molecule participating in various tumors. For example, miR-1307 was increased in prostate cancer which was able to promote prostate cancer cell growth through regulating ZEB1 [27]. Knockdown of miR-1307 inhibits proliferation and migration of gastric cancer cells through repressing SLC25A33 [28]. MiR-1307 confers tamoxifen resistance in breast cancer cells through downregulating SSTR2a/STAT3 axis [29]. Meanwhile, we found that the expression levels of miR-1307 -3p were up-regulated in CRC tissues by bioinformatic analysis (data not shown). However, the specific role of miR-1307 -3p and its molecular mechanisms in CRC remain unclear. 17: Here, this study aimed to investigate whether miR-1307 -3p might serve as a potential molecular target for CRC treatment by using in vitro gain and loss function assay. We showed that miR-1307 -3p promoted proliferation and invasion of CRC cells through binding with its downstream target AKT3. 18: ## Materials and methods 19: ### Collection of tissue samples 20: Forty-eight pairs of clinical tumor tissues and adjacent non-tumor tissues from CRC patients were obtained from the First People’s Hospital of Jinan (Jinan, China). All patients who participated in this study were diagnosed with primary CRC by histopathological examination. None of them received any treatment before surgery. All collected samples were immediately frozen in liquid nitrogen after resection for further detection. 21: ### Cell culture 22: Human normal colonic epithelium NCM460 cells and CRC cell lines HCT116, SW480, SW620, LoVo and DLD1 cells were obtained from the American Type Culture Collection (ATCC). All these cells were maintained in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) at 37 °C under a humidified atmosphere with 5% CO2. 23: ### Transfection 24: Recombinant lentiviruses carrying miR-negative control (miR-NC), miR-1307-3p mimic, miR-1307-3p inhibitor, AKT3 overexpression (AKT3 Oe) or si-AKT3 were purchased from GeneCopoeia (Guangzhou, China). Transfection was performed using PolyJet reagent (SignaGen Laboratories) according to the manufacturer’s protocol. 25: ### Cell counting kit 8 (CCK8) assay 26: The effect of miR-1307-3p on cell proliferation capability was detected using CCK8 assay. In brief, transfected HCT116 or SW480 cells (8 × 103 per well) were seeded into 96-well plates. At different time points (24 h, 48 h and 72 h), 10 µL CCK8 solution (KeyGEN Biotech) was added to each well and then incubated at 37 °C for 2 h. Absorbance at OD450 nm was optically measured using a microplate reader. 27: ### Cell invasion assay 28: Cell invasion potential was assessed using Matrigel invasion chambers (EMD Millipore). After transfection for 48 h, 2 × 104 cells resuspended in serum-free medium were seeded into the upper chambers pre-coated with Matrigel. The chambers were then placed into the lower chambers containing RPMI1640 medium with 10% FBS. After incubation at 37 °C for 48 h, invading cells on the lower surface were fixed with methanol and stained with crystal violet. The stained cells were counted under an inverted microscope (Leica). 29: ### qRT-PCR 30: Total RNA was extracted from tissues or cells using TRIzol reagent (Ambion) according to the manufacturer’s instructions. The expression levels of miR-NC or miR-1307-3p were detected using All-in-One™ miRNA qRT-PCR Detection Kit (Genecopoeia). The relative expression levels were calculated using 2−ΔΔCT method. GAPDH or U6 was used as endogenous control. 31: ### Western blot analysis 32: Proteins from tissues or cells were extracted using RIPA lysis buffer (Beyotime). An equal amount of protein samples were loaded to SDS-PAGE gels followed by transfer to PVDF membranes. The membranes were then incubated overnight at 4 °C with primary antibodies including AKT3 (1:5000; Abcam) and GAPDH (1:10,000; Abcam). The membranes were then washed three times before incubation with HRP-conjugated secondary antibodies at room temperature for 1 h. Protein bands were visualized using enhanced chemiluminescence reagent (GE Healthcare Life Sciences). 33: ### Luciferase reporter assay 34: Wild-type miR-1307-3p-binding sequences within AKT3 3′ UTR region or mutant sequences were inserted into pGL3-luciferase reporter vector (Promega). HCT116 or SW480 cells were co-transfected with miR-1307-3p mimic or miR-negative control along with AKT3 wild-type or mutant vectors. After transfection for 48 h, firefly and renilla luciferase activities were detected using Dual-Glo® Luciferase Assay System (Promega) according to manufacturer’s protocol. 35: ### Isolation of exosomes 36: Exosomes were isolated from conditioned media of CRC transfected with recombinant lentiviruses using Total Exosome Isolation Reagent from Cell Culture Media (Invitrogen) following the manufacturer's protocols. In brief, supernatants from cultured cells were centrifuged at 2000 g for 30 min at 4 °C to remove cell debris. The exosomes were then precipitated using Total Exosome Isolation Reagent at 4 °C for 30 min. After centrifugation at 10,000×g for 10 min at 4 °C, the pellet was dissolved in phosphate buffered saline (PBS) for further detection. 37: ### Transmission electron microscopy (TEM) 38: CRC-derived exosomes were fixed using 2.5% glutaraldehyde at 4 °C overnight. After wash with PBS three times, exosomes were dehydrated with alcohol series followed by embedding with resin overnight at room temperature. Ultrathin sections were imaged using TEM at 80 kv. 39: ### The internalization of exosomes into tumor cells 40: FITC-labeled CRC-derived exosomes were obtained after transfection with FITC-conjugated green fluorescent protein gene using Lipofectamine 2000 ™. HCT116 or SW480 cells were incubated with FITC-labeled exosomes for 12 h. After washing with PBS three times to remove free exosomes, Cells were analyzed using flow cytometry. 41: ### Statistical analysis 42: All experiments were performed independently at least three times. Data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism software version 6.0 (GraphPad Software Inc., San Diego, CA). The differences between groups were analyzed using Student’s t-test or ANOVA followed by Tukey test. P < 0.05 indicates statistical significance. 43: ## Results 44: ### MiR-1307-3p is up-regulated in CRC tissues and cells 45: To determine the precise role of miR-1307-3p in CRC progression, we first investigated its expression levels in clinical tumor samples and adjacent non-tumor tissues from CRC patients as well as normal colonic epithelium NCM460 cells and CRC cell lines HCT116, SW480,