| Objective:Renal cell carcinoma(RCC)originates from the renal parenchyma and is a common urological malignancy,with distant metastases found in 30%of patients at the early stage of diagnosis.RCC is less sensitive to radiotherapy and chemotherapy,so targeted drugs have become the main treatment modality for advanced RCC.Sunitinib is a multi-targeted tyrosine kinases inhibitor(TKI)that inhibits vascular endothelial growth factor(VEGF)receptors and platelet-derived growth factor(PDGF)receptors to effectively inhibit tumor angiogenesis,thereby inhibiting tumor proliferation and metastasis.However,in the case of significant therapeutic effects,targeted drugs are also subject to the problem of drug resistance in patients during treatment.Recent studies have revealed that pyroptosis plays an important role in the development of RCC,and alterations in the expression of related genes can be used as a biological marker to predict the prognosis of RCC.Long noncoding RNA(LncRNA)is an RNA longer than 200 nucleotides that is not directly involved in coding for proteins but plays an important regulatory role in the transcription and translation of protein-coding genes.LncRNAs have been demonstrated in several studies to be involved in the development of cancer,including tumor cell proliferation,invasion,metastasis,apoptosis and drug resistance,and can be used as an important biological indicator for early diagnosis of tumors and assessment of late prognosis.However,the role of pyroptosis-related LncRNA in sunitinib resistance in RCC is poorly understood.In summary,this study investigated the molecular biology of pyroptosisrelated LINC00941 in the progression of RCC and its molecular biology mechanism in the occurrence of sunitinib resistance to provide a novel molecular biology marker for clinical diagnosis of RCC and prediction of sunitinib efficacy.Methods:1.The bioinformatics software was used to analyze the expression of LncRNAs associated with pyroptosis in the RCC-KIRC dataset,and the results were used to further construct a clinical prognostic model for RCC and to analyze it in conjunction with clinically relevant indicators,and finally to screen out the LncRNAs with the most significant prognostic value and higher expression in RCC.2.The expression of LINC00941 in 72 cases of paraneoplastic normal tissues and 539 cases of tumor tissues in the TCGA-KIRC public dataset was analyzed by bioinformatics software to clarify the expression of LINC00941 in renal cell carcinoma and its relationship with tumor TNM stage,gender,tumor grade and prognostic survival.The expression of LINC00941 in RCC tumor tissues and paraneoplastic normal tissues was further verified by qRT-PCR in 20 patients who underwent radical nephrectomy in recent clinical years.3.We collected paraneoplastic normal and tumor tissue samples from 10 patients who underwent radical nephrectomy in recent years and further validated the expression of LINC00941 in RCC tumor tissues and paraneoplastic normal tissues using qRT-PCR.4.Stable expression cell lines were constructed by designing LINC00941-specific knockdown viruses(sh LINC00941#1 and sh LINC00941#2),specific overexpression viruses(LINC00941)and negative control viruses(sh NC),and infecting in vitro cultured RCC cell lines,786-O cells and ACHN cells with LINC00941 expression knockdown and overexpression using the specific viral infection solution.The knockdown and overexpression efficiency of the three viruses were examined by applying qRT-PCR.Subsequently,EdU assay,Transwell cell invasion assay in vitro,low-density clone formation assay and wound healing assay were used to examine the effects of knockdown and overexpression of LINC00941 on the tumor biological behavior of RCC cell lines 786-O cells and ACHN cells in vitro.5.786-O cells were treated and cultured using different concentrations of pyroptosis inhibitors,and the expression of LINC00941 in the treated cells was detected by qRTPCR method.Subsequently,the localization of LINC00941 in 786-O cells and ACHN cells was detected by fluorescence in situ hybridization.The pull-down proteins downstream of LINC00941 were further analyzed by RNA pull-down assay,the pulleddown proteins were analyzed by protein mass spectrometry identification assay,and validated by Western-blot assay.6.16 male BALB/c nude mice of the same week of age were randomly divided into 4 groups of 4 mice each.A xenograft tumor model was constructed using the preconstructed LINC00941,sh LINC00941#1,sh LINC00941#2 and sh NC of the 786-O stable transfer cell line by subcutaneous injection behind the neck of the nude mice,and the tumor growth volume was measured every 4 days after the construction and the curve of tumor growth was plotted.After 6 weeks of tumor growth,the proliferation of 786-O cells in different treatment groups was examined in vivo by IVIS small animal live imaging assay.Subsequently,nude mice were executed using the decapitation method to observe the tumor growth size in different groups,and the tumor mass was weighed,and the expression of Ki67 and hnRNPM in tumor tissues was examined using immunohistochemistry assays.7.20 male BALB/c nude mice of the same week of age were randomly divided into four groups of five each,and tumor lung metastasis models were constructed by tail vein injection of 786-O stable transgenic cells with different treatments.After the 6-week lung metastasis model was successfully constructed,the lung metastases of different groups were examined using IVIS small animal live imaging experiments.The nude mice were subsequently executed by decortication,and the lungs were dissected and removed from the nude mice to observe the lung tumor metastasis in different groups.The lung metastasis model tissues were stained by hematoxylin-eosin staining to observe the tumor metastasis growth in the lung tissue samples of different groups.8.Transcriptome sequencing analysis of 786-O cells overexpressing LINC00941 as well as negative control sh NC was performed to explore the downstream related regulatory pathways of LINC00941 in RCC and validate the related pathways using Western-blot experiments.9.Sunitinib was added to 786-O cell medium using a low concentration gradient method and co-cultured with the cells to eventually establish a sunitinib-resistant 786-O-R cell line.LINC00941-specific knockdown virus(sh LINC00941#1 and sh LINC00941#2),specific overexpression virus(LINC00941)and negative control virus(sh NC)were used to transfect the 786-O-R cell line in vitro to construct a stable trans-resistant cell line.The knockdown and overexpression efficiency of the three viruses were examined by qRTPCR.The semi-inhibitory concentration IC50 of sunitinib in sensitive and resistant cell lines was determined using cytotoxicity assay.10.Sixteen same-week-old male BALB/c nude mice were randomly divided into four groups of four mice each.A xenograft tumor model was constructed by subcutaneous injection of sunitinib-sensitive 786-O stable-transformed cell line and sunitinib-resistant 786-O-R stable-transformed cell line overexpressing LINC00941 in the posterior neck of nude mice.After the tumors were visible subcutaneously for one-week,different groups of nude mice were treated with sunitinib by gavage every 2 days,and the tumor volume size was measured and recorded every 4 days for about 4 weeks.The growth of 786-O cells of different treatment groups in vivo after drug treatment was detected by IVIS small animal live imaging assay.Subsequently,nude mice were executed by decortication,tumor growth size was observed in different groups,tumor mass was weighed,and Ki67 and hnRNPM expression in tumor tissues were detected using immunohistochemistry assays.Results.1.By screening the TCGA-KIRC dataset for pyroptosis-related LncRNAs,the final 12 differentially expressed and pyroptosis-related LncRNAs were identified.The clinical prognostic model was constructed by 12 LncRNAs,and the results showed that our model had a more reliable but accurate predictive performance for the clinical prognosis of RCC patients and was better than the LncRNA prognostic models published in recent years.We further constructed the clinical prognostic model as nomogram to predict the prognosis of patients more accurately.The results showed that the clinical nomogram was also accurate and reliable in assessing the prognosis of patients.2.The expression of LINC00941 was found to be significantly higher in RCC tumor tissues than in normal tissues adjacent to cancer(P<0.05),and the expression level of LINC00941 was positively correlated with poor TNM stage,tumor grade and prognostic survival of patients(P<0.05).The constructed prognostic survival column line graph suggested that LINC00941 could be used as a reliable biological marker to predict the prognosis of patients.3.By interfering with the expression level of LINC00941,the proliferation,invasion and migration levels of RCC cells can be inhibited in vitro.In contrast,promoting the expression level of LINC00941 enhances the tumor biological behaviors such as proliferation,invasion and migration levels of RCC cells.4.The expression of LINC00941 was significantly inhibited by the focal death inhibitor VX765,further validating the previous bioinformatics results.The fluorescence in situ hybridization technique showed that LINC00941 was significantly expressed in both cytoplasm and nucleus,indicating that LINC00941 has an important role in the development of RCC.The RNA pull-down assay and protein profiling revealed that LINC00941 was positively associated with hnRNPM protein gene.5.The results of animal experiments showed that interfering with the expression of LINC00941 helped to inhibit the volume and rate of tumor growth in vivo,while promoting the expression of LINC00941 helped to promote the volume and rate of tumor growth in vivo.In addition,immunohistochemical experiments in nude mice tumor tissues further verified that the expression levels of LINC00941 were positively correlated with the expression levels of Ki67 and hnRNPM.6.The results of animal experiments showed that interfering with the expression of LINC00941 helped to suppress lung tumor metastasis in vivo,while promoting the expression of LINC00941 helped to promote lung tumor metastasis in vivo.In addition,the positive correlation between the expression level of LINC00941 and tumor metastasis in lung tissue of nude mice was further verified by using hematoxylin-eosin staining.7.Transcriptome sequencing analysis and Western-blot experiments revealed that LINC00941 plays a key role in the development of RCC mainly through regulating the NOD-like receptor signaling pathway.8.Increasing the expression level of LINC00941 in vitro leads to an increase in cellular semi-inhibitory IC50 concentration to sunitinib and promotes the development of resistance to sunitinib drug in RCC-sensitive cell lines.9.The results of animal experiments showed that increasing the expression level of LINC00941 in vivo diminished the therapeutic effect of tumors on sunitinib,increased drug resistance,and positively correlated with the expression levels of Ki67 and hnRNPM.Conclusion:We screened 12 LncRNAs with clinical prognostic significance associated with pyroptosis through TCGA database,and constructed a clinical prognostic model and prognostic nomogram to reliably predict the prognosis of RCC patients.Among the 12 LncRNAs,LINC00941 was further screened for the most significant one with clinical prognosis.LINC00941 is highly expressed in RCC tumor tissues and is involved in tumor proliferation,invasion,and migration in vivo and vitro,mainly through the regulation of hnRNPM protein and NOD-like receptor signaling pathway.LINC00941 may be a biological marker and therapeutic target for predicting tumor prognosis and the efficacy of sunitinib therapy. |
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