LAMC2 regulates proliferation, migration, and invasion mediated by the Pl3K/AKT/mTOR pathway in oral

Abstract

Background: Oral squamous cell carcinoma (OSCC) is a common malignant tumor. Recently, Laminin Gamma 2 (LAMC2) has been shown to be abnormally expressed in OSCC; however, how LAMC2 signaling contributes to the occurrence and development of OSCC and the role of autophagy in OSCC has not been fully explored. This study aimed to analyze the role and mechanism of LAMC2 signaling in OSCC and the involvement of autophagy in OSCC.

Methods: To explore the mechanism by which LAMC2 is highly expressed in OSCC, we used small interfering RNA (siRNA) to knock down LAMC2 to further observe the changes in the signaling pathway. Furthermore, we used cell proliferation assays, Transwell invasion assays, and wound-healing assays to observe the changes in OSCC proliferation, invasion, and metastasis. RFP-LC3 was used to detect the level of autophagy intensity. A cell line-derived xenograft (CDX) model was used to detect the effect of LAMC2 on tumor growth in vivo.

Results: This study found that the level of autophagy was correlated with the biological behavior of OSCC. The downregulation of LAMC2 activated autophagy and inhibited OSCC proliferation, invasion, and metastasis via inhibiting the PI3K/AKT/mTOR pathway. Moreover, autophagy has a dual effect on OSCC, and the synergistic downregulation of LAMC2 and autophagy can inhibit OSCC metastasis, invasion, and proliferation via the PI3K/AKT/mTOR pathway.

Conclusions: LAMC2 interacts with autophagy to regulate OSCC metastasis, invasion, and proliferation via the PI3K/AKT/mTOR pathway. LAMC2 down-regulation can synergistically modulate autophagy to inhibit OSCC migration, invasion, and proliferation.

Keywords: 3-Methyladenine; Autophagy; LAMC2; OSCC; PI3K/AKT/mTOR pathway; Rapamycin.

Figure 1. LAMC2 is highly expressed in HNSC and OSCC cell lines. (A–C) The GEPIA database shows the level of LAMC2 expression in different tumors. The levels of LAMC2 expression were abnormally elevated in HNSC patients compared to that of normal tissues (tumor tissues, n = 519; normal tissues, n = 44). (D) Patients with high and low LAMC2 expression display different survival curves. The survival rate was significantly reduced in patients with high LAMC2 expression compared to patients with low LAMC2 expression tumors. (E, F) LAMC2 expression in HNOEC and different OSCC cell lines. Each experiment was conducted at least three times and at least three sets of data were collected from each experiment for statistical analysis (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 2. LAMC2 downregulation suppresses cell proliferation, metastasis, and invasion of OSCC tumors and inhibits the occurrence of EMT. (A, B) WB and RT-qPCR shows the level of LAMC2 protein and mRNA expression, respectively, following a LAMC2 knockdown using siRNA in cal-27 cells. (C, D) Wound healing assays (Original magnification, ×50) and Transwell invasion (Original magnification: ×100) were used to observe the ability of cal-27 cells metastasis and invasion ability after an LAMC2 knockdown using siRNA. Compared with the si-NC group, cell migration and invasion was significantly decreased in the si-LAMC2 group. (D, A) WB was used to observe the changes in EMT progression in cal-27 cells following an LAMC2 knockdown using siRNA. Similarly, the EMT process in the si-LAMC2 group significantly differed from that in the si-NC group. (E–G) To observe the changes in cal-27 cell viability and proliferation after an LAMC2 knockdown, we used a clone formation assay and cell proliferation assays. The results were consistent with the above experiments, and cell proliferation and viability were significantly decreased in the si-LAMC2 group. Each experiment was conducted at least three times and at least three sets of data were collected from each experiment for statistical analysis (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 3. Silencing LAMC2 can activate autophagy through the PI3K/AKT/mTOR pathway. (A) After a knockdown of LAMC2, the changes in the proteins downstream of LAMC2 were displayed by WB. The level of PI3K, AKT, and mTOR protein phosphorylation was significantly decreased, and autophagy-related proteins (LC3, Beclin-1) were activated. B–C show the change in the level of autophagy following LAMC2 downregulation using an MDC staining assay and RFP-LC3. In the si-LAMC2 group, both experiments exhibited increased levels of autophagy. Each experiment was conducted at least three times and at least three sets of data were collected from each experiment for statistical analysis (*p < 0.05; **p < 0.01; ***p < 0.001, NS, not significant).

Figure 4. Inhibition or activation of autophagy can inhibit OSCC. (A) A CCK-8 assay was performed to research the effects of different concentrations of 3-Methyladenine and Rapamycin on cell viability. Compared with the control group, different drug concentrations of 3-Methyladenine (0.25, 0.5, 1, 3 mMOL/L) and Rapamycin (1, 10, 20, and 30 μMOL/L) inhibited the viability of Cal-27 cells. (B, F) The changes in autophagy-related proteins and EMT-related proteins were detected by WB after the addition of various drugs. After the addition of 3-Methyladenine (0.5 mMOL/L) and Rapamycin (20 μMOL/L), EMT, and autophagy-related proteins showed corresponding changes. (C–E) The changes in the biological behavior of CAL-27 cells were detected by the Wound healing assays (original magnification: ×50), Transwell invasion (original magnification: ×100), and colony formation assay after the addition of drugs (3-Methyladenine (0.5 mMOL/L) and Rapamycin (20 μMOL/L)). (G–H) show the change in the level of autophagy after the addition of agents (3-Methyladenine (0.5 mMOL/L) and Rapamycin (20 μMOL/L)). The intensity of autophagy increased following the addition of 3-Methyladenine (0.5 mMOL/L). After the addition of Rapamycin (20 μMOL/L), the autophagy intensity was decreased. Each experiment was conducted at least three times and at least three sets of data were collected from each experiment for statistical analysis (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 5. The increased level of autophagy in OSCC induced by LAMC2 silencing was reversed by 3-Methyladenine. (A) After a knockdown of LAMC2 and addition of various drugs (3-Methyladenine (0.5 mMOL/L) and Rapamycin (20 μMOL/L)), the changes in the proteins downstream of LAMC2 were evaluated by WB. The levels of PI3K, AKT, and mTOR protein phosphorylation was significantly decreased in the si-LAMC2 group. Some autophagy-related proteins (LC3, Beclin-1) were activated and other autophagy related proteins (P62) were inhibited. In the si-LAMC2+3MA group, the phosphorylation level of the PI3K, AKT, and mTOR protein signaling pathway and autophagy-related proteins (LC3, Beclin-1, and P62) returned to normal levels. (B, C) show the changes in the level of autophagy following LAMC2 downregulation and the addition of drugs (3-Methyladenine (0.5 mMOL/L) and Rapamycin (20 μMOL/L)) by MDC Staining Assay and RFP-LC3. The results were consistent with the protein results. Each experiment was conducted at least three times and at least three sets of data were collected from each experiment for statistical analysis (*p < 0.05; **p < 0.01; ***p < 0.001, NS, not significant).

Figure 6. The inhibitory effect of LAMC2 downregulation on OSCC was reversed by 3-Methyladenine. (A–C) Changes in the biological behavior of Cal27 cells were detected by wound healing assays (original magnification: ×50), Transwell invasion (original magnification: ×100) and clone formation assay after the knockdown of LAMC2 and addition of drugs (3-Methyladenine (0.5 mMOL/L) and Rapamycin (20 μMOL/L). (D) WB shows the changes in EMT-related proteins after a knockdown of LAMC2 and addition of drugs (3-Methyladenine (0.5 mMOL/L) and Rapamycin (20 μMOL/L)). Each experiment was conducted at least three times and at least three sets of data were collected in each experiment for statistical analysis (*p < 0.05; **p < 0.01; ***p < 0.001, NS, not significant).

Figure 7. Silencing LAMC2 can increase the level of autophagy in OSCC and inhibit OSCC growth in vivo. (A) Tumors were excised from mice and the curve for subcutaneous tumor growth volume was calculated. (B) WB shows the level of different protein expression in vivo after a LAMC2 knockdown (three mice were selected from each group). Representative HE staining and IHC results are presented in (C, D). (E) Three sets (Control, si-NC, si-LAMC2) of TEM images (autophagosomes are marked with) (Original magnification: ×2000 and ×5000). Each experiment was conducted at least three times and at least three sets of data collected for statistical analysis for each experiment (*p < 0.05; **p < 0.01; ***p < 0.001, NS, not significant).

Figure 8. A schematic diagram of LAMC2, autophagy, and the PI3K/AKT/mTOR pathway in OSCC.