Application of C1QTNF1-ASI / miR-346-LDHA / PDK regulatory axis in diagnosis and treatment of osteosarcoma

Abstract

The invention belongs to the technical field of diagnosis and treatment of osteosarcoma, and particularly relates to application of a C1QTNF1-AS1 / miR-346-LDHA / PDK regulation axis in diagnosis and treatment of osteosarcoma, a full-chain technical system of'diagnostic marker-double-target intervention-clinical transformation 'is constructed on the basis of the C1QTNF1-AS1 / miR-346-LDHA / PDK regulation axis, and accurate diagnosis and metabolic intervention of osteosarcoma are realized through multi-dimensional experimental verification; in the diagnosis, a C1QTNF1-AS1 / miR-346 combined detection kit based on qPCR is applied to early screening of osteosarcoma; according to the treatment application, the C1QTNF1-AS1 adenovirus and the miR-346 lipidosome are used in a combined manner, and LDHA and PDK1 are synchronously inhibited, so that the Warburg effect related metabolic flux is intervened.

Description

Technical Field

[0001] This invention belongs to the field of diagnosis and treatment technology of osteosarcoma, specifically the application of the C1QTNF1-AS1 / miR-346-LDHA / PDK regulatory axis in the diagnosis and treatment of osteosarcoma. Background Technology

[0002] Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. It is highly malignant and metastasizes early. The 5-year survival rate of patients has long been stagnant at around 60%. 30% to 40% of patients relapse and metastasize within 2 years. The key reason is that the Warburg effect of tumor cells leads to drug resistance and enhanced invasion.

[0003] The diagnosis of osteosarcoma mainly relies on X-ray, CT, MRI and pathological biopsy, which makes it difficult to detect small lesions in the early stage and lacks high-specificity molecular markers; serum ALP and LDH are commonly used indicators, but they can also be elevated in benign bone diseases, so they are not specific enough.

[0004] Standard treatment relies on surgery and high-dose chemotherapy (cisplatin, doxorubicin, methotrexate), but cisplatin and other drugs have dose-limiting toxicities.

[0005] However, the development of targeted drugs is lagging behind, and existing drugs mostly focus on a single target. For example, although the LDHA inhibitor GNE-140 can inhibit lactate production, clinical trials show that its monotherapy effect is limited and it is easy to induce tumor cells to maintain glycolysis through compensatory pathways such as PDK1.

[0006] Although the PDK1 inhibitor dichloroacetic acid (DCA) can promote the entry of pyruvate into the tricarboxylic acid cycle, it cannot cross the blood-brain barrier and is highly toxic to normal cells.

[0007] In summary, current diagnostic technologies lack highly specific and early-detectable biomarkers to pinpoint microfoci, while treatment is limited by single-target metabolic compensation and dose toxicity, making it impossible to achieve sustained tumor suppression within safe boundaries. Summary of the Invention

[0008] The purpose of this invention is to provide the application of the C1QTNF1-AS1 / miR-346-LDHA / PDK regulatory axis in the diagnosis and treatment of osteosarcoma, so as to solve the above-mentioned technical problems.

[0009] This invention proposes the use of miR-346 in the preparation of medicaments for the treatment of osteosarcoma, for the simultaneous inhibition of LDHA and PDK1 to intervene in aerobic glycolysis of tumor cells.

[0010] Furthermore, it can be used in combination with chemotherapy / radiotherapy.

[0011] This invention also proposes the application of C1QTNF1-AS1 in upregulating miR-346 in osteosarcoma cells.

[0012] This invention also proposes a method for in vitro detection of osteosarcoma, which uses real-time quantitative PCR to detect the expression levels of C1QTNF1-AS1 and miR-346 in the tested in vitro samples to determine the correlation between the tested samples and osteosarcoma.

[0013] This invention also proposes the application of a combined detection kit for C1QTNF1-AS1 and miR-346 in the early screening of osteosarcoma.

[0014] The present invention also proposes a pharmaceutical composition for inhibiting glycolysis-related metabolism in osteosarcoma, the composition comprising a combination of C1QTNF1-AS1 adenovirus and miR-346 liposomes.

[0015] The present invention also proposes the use of a pharmaceutical composition comprising C1QTNF1-AS1 adenovirus and miR-346 liposomes in adjuvant therapy after osteosarcoma surgery to reduce the recurrence rate.

[0016] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0017] I. This invention achieves highly accurate diagnosis by detecting the expression level of C1QTNF1-AS1 / miR-346.

[0018] II. This invention achieves "synergistic metabolic blockade" by simultaneously inhibiting LDHA and PDK1 through the C1QTNF1-AS1 / miR-346 axis:

[0019] Third, this invention is the first to demonstrate that C1QTNF1-AS1, as an upstream regulator of miR-346, intervenes in the metabolic network through a "sponge adsorption-dual-target gene silencing" mechanism. miR-346 simultaneously targets LDHA and PDK1 (a key rate-limiting enzyme in glycolysis), while C1QTNF1-AS1 amplifies the inhibitory effect on metabolic pathways by enhancing miR-346 activity, forming a "LncRNA-miRNA-dual-target gene" regulatory model, providing a new paradigm for the application of non-coding RNA in tumor metabolism.

[0020] Fourth, this invention achieves “marker-target” linkage through the same regulatory axis. The C1QTNF1-AS1 / miR-346 combined detection kit based on qPCR can be used for early screening. The combined use of C1QTNF1-AS1 adenovirus and miR-346 liposomes can be used as an adjuvant therapy after surgery to reduce the recurrence rate. Both have been verified in animal experiments and have the potential for rapid translation. Attached Figure Description

[0021] Figure 1To analyze the differential expression between osteosarcoma and osteoblasts and to validate the low expression of C1QTNF1-AS1;

[0022] Figure 2 The effects of C1QTNF1-AS1 overexpression on osteosarcoma cell growth and metabolism and xenograft tumor growth;

[0023] Figure 3 Prediction, dual-fluorescence validation, and expression association of C1QTNF1-AS1–miR-346 interaction;

[0024] Figure 4 The downregulation of miR-346 promotes the proliferation, migration, invasion, glucose metabolism, and tumorigenesis of osteosarcoma cells.

[0025] Figure 5 A rescue experiment to partially reverse the effects of miR-346 knockdown on C1QTNF1-AS1 in inhibiting osteosarcoma and inhibiting glycolysis;

[0026] Figure 6 To validate miR-346's direct targeting of LDHA and PDK1 (dual fluorescence, qPCR, and protein detection).

[0027] Figure 7 To investigate the regulatory relationship between C1QTNF1-AS1 and LDHA and PDK1 downregulation via miR-346 and to conduct experiments on its recovery;

[0028] Figure 8 This is a technical research roadmap for the application of the regulatory axis proposed in this invention in the diagnosis and treatment of osteosarcoma. Detailed Implementation

[0029] The following examples use the C1QTNF1-AS1 / miR-346-LDHA / PDK regulatory axis as the core technical pathway to illustrate its application in the diagnosis and / or treatment of osteosarcoma.

[0030] The embodiments described above are used to illustrate the technical solutions of the present invention and are intended to help understand the spirit and essence of the present invention. They do not constitute a limitation on the scope of protection. Without departing from the concept of the present invention and subject to the claims, those skilled in the art can make equivalent substitutions or conventional optimizations to the detection target, carrier type, compatibility excipients, route of administration, applicable population, and combination therapy strategies based on conventional molecular biology, cell biology, and pharmaceutical engineering methods. All such substitutions and optimizations should be covered within the scope of the present invention. Unless otherwise stated, the term "comprising / including" is an open-ended expression, and "consisting essentially of..." is a semi-closed-ended expression; parameter ranges such as "X–Y" include endpoints; quantitative expression can be in the form of relative quantification or normalized expression.

[0031] The embodiments of the present invention are based on the C1QTNF1-AS1 / miR-346-LDHA / PDK1 regulatory axis, constructing a full-chain technology system of "diagnostic biomarkers - dual-target intervention - clinical translation", and realizing the accurate diagnosis and metabolic intervention of osteosarcoma through multi-dimensional experimental verification.

[0032] The diagnostic application uses a qPCR-based C1QTNF1-AS1 / miR-346 combined detection kit for early screening of osteosarcoma; the therapeutic application uses a combination of C1QTNF1-AS1 adenovirus and miR-346 liposomes to simultaneously inhibit LDHA and PDK1, thereby intervening in Warburg effect-related metabolic fluxes.

[0033] Specifically, real-time quantitative PCR was used to detect the expression levels of C1QTNF1-AS1 and miR-346 in the tested in vitro samples to determine the correlation between the tested samples and osteosarcoma.

[0034] The kit is based on qPCR and is used to detect the expression levels of C1QTNF1-AS1 and miR-346 simultaneously to obtain joint discrimination results.

[0035] To identify differentially expressed genes in osteosarcoma cells, this invention uses the Sanger platform to analyze the gene expression profiles in the GSE126209 dataset extracted from the GEO database.

[0036] Using the limma package (version 4.4.0) in R, genes that show significant differences between osteosarcoma cells and normal osteoblasts were screened based on the criteria of log fold change (FC) > 1 and p < 0.05.

[0037] Visualize these findings using volcano maps and heatmaps. Figure 1 (A and 1B).

[0038] The results showed that the expression level of C1QTNF1-AS1 in osteosarcoma cells was significantly lower than that in normal osteoblasts. Figure 1 C). This invention used qPCR to detect the expression level of C1QTNF1-AS1 in osteoblasts (HFOB1.19) and various osteosarcoma cell lines (including Saos-2, MG63, HOS and U2OS).

[0039] It is noteworthy that, compared to normal osteoblast cell lines, the expression level of C1QTNF1-AS1 was significantly reduced in osteosarcoma cell lines, with the most significant decrease observed in the MG63 and U2OS cell lines. Figure 1 D).

[0040] The present invention also proposes a pharmaceutical composition for inhibiting osteosarcoma glycolysis, comprising C1QTNF1-AS1 adenovirus or its combination with miR-346 liposomes;

[0041] To elucidate the specific function of C1QTNF1-AS1 in osteosarcoma cells, this invention constructed cell lines overexpressing C1QTNF1-AS1 (lnc-mim) and a negative control (mim-NC).

[0042] Through CCK-8 proliferation experiment ( Figure 2 A) Scratch healing test ( Figure 2 B and 2C) and trans-pore invasion test ( Figure 2 The results (D and 2E) showed that overexpression of C1QTNF1-AS1 significantly inhibited the proliferation, migration and invasion of MG63 and U2OS cells.

[0043] Given the important role of the Warburg effect in tumor growth, this invention evaluated the ECAR (energy consumption rate) and OCR (oxidative phosphorylation rate) of osteosarcoma cells in the C1QTNF1-AS1 overexpression group and the control group through metabolic flux analysis, which reflect glycolytic activity and oxidative phosphorylation level, respectively.

[0044] The results showed that overexpression of C1QTNF1-AS1 significantly reduced the ECAR-R in MG63 and U2OS cells. Figure 2 F), while enhancing its OCR ( Figure 2 G).

[0045] Furthermore, this overexpression significantly reduced ATP production in these cells. Figure 2 H) and lactic acid secretion ( Figure 2 I), while the glucose level in the culture supernatant was significantly increased ( Figure 2 J) indicates a decrease in glucose consumption.

[0046] To verify the anti-cancer effect of C1QTNF1-AS1 in vivo, this invention conducted xenograft experiments in nude mouse models by subcutaneously injecting either stably overexpressing C1QTNF1-AS1 or control MG63 cells. Compared with the control group, the overexpression group exhibited a slower tumor growth rate and a smaller tumor burden. Figure 2 K-2M).

[0047] Immunohistochemical staining further confirmed that Ki67 expression was reduced in xenografts of the C1QTNF1-AS1 overexpression group. Figure 2 These results (N and 2O) collectively demonstrate that C1QTNF1-AS1 can inhibit osteosarcoma cell proliferation, migration, invasion, and the Warburg effect both in vitro and in vivo.

[0048] To investigate the regulatory role of C1QTNF1-AS1 in aerobic glycolysis in osteosarcoma, this invention uses TargetScan to predict miRNAs that may bind to C1QTNF1-AS1, PDK1, PDK2, PDK3 and LDHA.

[0049] Cross-analysis revealed that miRNAs such as miR-346 are functionally associated with aerobic glycolysis. Figure 3 A). Based on the miRDB and miRanda algorithms, this invention predicts the binding site of C1QTNF1-AS1 to miR-346 and designs mutant sequences for verification.

[0050] Dual-luciferase reporter system experiments showed that, compared with the control group, co-transfection with wild-type C1QTNF1-AS1 significantly enhanced luciferase activity with miR-346 mimics. However, this effect was completely eliminated upon mutation of the binding site. Figure 3 B) confirmed that miR-346 and C1QTNF1-AS1 directly interact at the predicted site. Figure 3 C).

[0051] To further elucidate the relationship between C1QTNF1-AS1-miR-346 and osteosarcoma cells, this invention established cell lines that stably overexpress C1QTNF1-AS1 (Lnc-mim) and control (mim-NC).

[0052] qPCR analysis showed that C1QTNF1-AS1 overexpression significantly upregulated miR-346 expression levels in osteosarcoma cells. Figure 3 D).

[0053] This invention proposes the use of miR-346 in osteosarcoma, in which miR-346 liposomes are administered via tail vein / intratumoral / peritoneal routes to inhibit the Warburg effect in osteosarcoma cells.

[0054] To investigate the function of miR-346 in osteosarcoma cells, we constructed a stable miR-346 knockdown cell line (si-miR) and a negative control (si-NC).

[0055] CCK-8 proliferation assays showed that decreased miR-346 expression significantly promoted osteosarcoma cell proliferation. Figure 4 A). The scratch healing assay results showed that reduced miR-346 expression enhanced the migration ability of osteosarcoma cells. Figure 4 B and 4C).

[0056] Transwell invasion assays further confirmed that miR-346 knockdown significantly enhanced the invasive ability of osteosarcoma cells. Figure 4 (D and 4E).

[0057] Metabolic analysis showed that miR-346 knockdown increased the level of ECAR, a biomarker of glycolysis, in MG63 and U2OS cell lines. Figure 4 F), while reducing the oxidative phosphorylation index OCR (F). Figure 4 G).

[0058] By detecting ATP levels, lactate production, and glucose consumption in cell supernatant ( Figure 4 H~4J) found that miR-346 inhibited the Warburg effect in osteosarcoma cells.

[0059] Xenograft experiments have demonstrated that, compared to the control group, a significant decrease in miR-346 significantly accelerates tumor growth and increases tumor burden. Figure 4 K-4M).

[0060] Immunohistochemical analysis showed that Ki67 expression was enhanced in miR-346-deficient xenograft tumors. Figure 4 (N and 4O). In summary, these findings indicate that miR-346 effectively inhibits the proliferation, migration, and aerobic glycolysis of osteosarcoma cells both in vitro and in vivo.

[0061] To investigate whether C1QTNF1-AS1 inhibits osteosarcoma (OS) development by targeting miR-346, we conducted rescue experiments using U2OS and MG63 cell lines.

[0062] By comparing CCK-8 proliferation experiments ( Figure 5 A) Scratch healing test ( Figure 5 B and 5C) and transwell invasion test ( Figure 5 (D and 5E) found that C1QTNF1-AS1 overexpression significantly inhibited the proliferation, migration, and invasion of osteosarcoma cells. However, knockdown of miR-346 partially alleviated this effect.

[0063] Metabolic profiling analysis showed that C1QTNF1-AS1 overexpression reduced ECAR (electro-associated cytokines) in MG63 and U2OS cells. Figure 5 F), and added OCR (F). Figure 5 G).

[0064] Quantitative analysis of ATP production, lactate secretion and glucose consumption in cell supernatant ( Figure 5 (H-5J) indicates that C1QTNF1-AS1 overexpression can alleviate the Warburg effect in osteosarcoma cells. Notably, knockdown of miR-346 can partially reverse glycolytic inhibition.

[0065] Validation using a xenograft model revealed that knocking down miR-346 counteracts the anti-tumor effect of C1QTNF1-AS1 overexpression, specifically manifested in tumor morphology ( Figure 5 K), volume ( Figure 5 L) and weight ( Figure 5 Changes in M), and immunohistochemical staining showing decreased Ki67 expression ( Figure 5 N and 5O). C1QTNF1-AS1 effectively inhibits the proliferation, migration, invasion, and Warburg effect of osteosarcoma cells both in vitro and in vivo by regulating miR-346-mediated glycolytic reprogramming.

[0066] The miRWalk algorithm predicts the binding sites of LDHA, PDK1, and miR-346, and then designs mutant sequences.

[0067] Dual luciferase assays showed that the miR-346 mimic could bind to wild-type LDHA and PDK1, resulting in a significant decrease in luciferase activity compared to the control group (NC).

[0068] However, by modifying the binding site, miR-346 mimics can no longer affect luciferase activity. Figure 6 A and 6B). This confirms that miR-346 specifically binds to LDHA and PDK1 at this site (A and 6B). Figure 6 C and 6D).

[0069] Researchers successfully established stable cell lines with miR-346 knockdown (si-miR) and a control group (si-NC). Quantitative PCR (qPCR) Figure 6 E) and Western blot analysis ( Figure 6 (F and 6G) ​​showed that miR-346 knockdown significantly upregulated the expression levels of PDK1 and LDHA in osteosarcoma cells.

[0070] To investigate the interaction between C1QTNF1-AS1 (lnc-mim) and miR-346, LDHA and PDK1, we established osteosarcoma cell lines that stably overexpress C1QTNF1-AS1 (lnc-mim) and negative control (mim-NC).

[0071] qPCR and Western blot analysis revealed that overexpression of C1QTNF1-AS1 significantly reduced the levels of LDHA and PDK1 in osteosarcoma cells. Figure 7 (A~7C).

[0072] Such as qPCR ( Figure 7 D) and Western blot analysis ( Figure 7As shown in E and 7F), our recovery experiments demonstrate that knocking down miR-346 can partially alleviate the decrease in LDHA and PDK1 levels caused by C1QTNF1-AS1 overexpression.

[0073] The above experiments confirm that C1QTNF1-AS1-miR-346 can simultaneously target two consecutive rate-limiting enzymes in the pyruvate-lactic acid metabolic pathway—creatine phosphate dehydrogenase 1 (PDK1) and lactate dehydrogenase A (LDHA). This dual inhibition creates a synergistic metabolic blockade effect, which is verified by the significant reduction in lactate production and ATP consumption in cells overexpressing miR-346. This strategy overcomes the inherent compensatory limitations of glycolysis and provides a novel intervention framework for the treatment of osteosarcoma.

[0074] This invention identifies C1QTNF1-AS1 and miR-346 as biomarkers for the early diagnosis of osteosarcoma. Analysis of their expression levels enables early detection and timely intervention.

[0075] Osteosarcoma cells possess aerobic glycolytic properties, and this invention proposes three treatment strategies:

[0076] 1. By regulating lactate dehydrogenase (LDHA) and creatine phosphate dehydrogenase 1 (PDK1), miR-346 function is restored to inhibit tumor growth;

[0077] 2. Targeting the LDHA / PDK1 complex disrupts the metabolic adaptation of osteosarcoma;

[0078] 3. Combining miR-346 targeted therapy with chemotherapy / radiotherapy can enhance efficacy.

[0079] The C1QTNF1-AS1-miR-346 axis, as the first confirmed dual inhibitor, simultaneously acts as a prognostic indicator and a therapeutic target in osteosarcoma metabolism.

[0080] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention; the actual structure is not limited thereto. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the invention, such designs should fall within the protection scope of the present invention.

Claims

1. Use of miR-346 in the preparation of a drug for treating osteosarcoma, for simultaneously inhibiting LDHA and PDK1 to intervene in the aerobic glycolysis of tumor cells.

2. Use of miR-346 according to claim 1 for the preparation of a medicament for the treatment of osteosarcoma, characterized in that, Combined with chemotherapy / radiotherapy.

3. Use of C1QTNF1-AS1 for up-regulating miR-346 in osteosarcoma cells.

4. A method for the in vitro detection of osteosarcoma, characterized in that, Real-time fluorescent quantitative PCR is used to detect the expression levels of C1QTNF1-AS1 and miR-346 in the sample of the subject, to determine the correlation between the sample and osteosarcoma.

5. Use of a C1QTNF1-AS1 and miR-346 combined detection kit in early screening of osteosarcoma.

6. A pharmaceutical composition for inhibiting glycolysis-related metabolism of osteosarcoma, the composition comprising a combined use of C1QTNF1-AS1 adenovirus and miR-346 liposome.

7. Use of a pharmaceutical composition comprising C1QTNF1-AS1 adenovirus and miR-346 liposome in reducing the recurrence rate in postoperative adjuvant therapy of osteosarcoma.

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