The human vault RNA enhances tumorigenesis and chemoresistance through the lysosome in hepatocellular carcinoma

The small non-coding <i>VTRNA1-1</i> (vault RNA 1–1) is known to confer resistance to apoptosis in several malignant cell lines and to also modulate the macroautophagic/autophagic flux in hepatocytes, thus highlighting its pro-survival role. Here we describe a new function of <i>VTRNA1-1</i> in regulating <i>in vitro</i> and <i>in vivo</i> tumor cell proliferation, tumorigenesis and chemoresistance. Knockout (KO) of <i>VTRNA1-1</i> in human hepatocellular carcinoma cells reduced nuclear localization of TFEB (transcription factor EB), leading to a downregulation of the coordinated lysosomal expression and regulation (CLEAR) network genes and lysosomal compartment dysfunction. We demonstrate further that impaired lysosome function due to loss of <i>VTRNA1-1</i> potentiates the anticancer effect of conventional chemotherapeutic drugs. Finally, loss of <i>VTRNA1-1</i> reduced drug lysosomotropism allowing higher intracellular compound availability and thereby significantly reducing tumor cell proliferation <i>in vitro</i> and <i>in vivo</i>. These findings reveal a so far unknown role of <i>VTRNA1-1</i> in the intracellular catabolic compartment and describe its contribution to lysosome-mediated chemotherapy resistance. <b>Abbreviations:</b> ATP6V0D2: ATPase H+ transporting V0 subunit d2; BafA: bafilomycin A1; CLEAR: coordinated lysosomal expression and regulation; CQ: chloroquine; DMSO: dimethyl sulfoxide; GST-BHMT: glutathionine S-transferase N-terminal to betaine–homocysteine S-methyltransferase; HCC: hepatocellular carcinoma; LAMP1: lysosomal associated membrane protein 1; LLOMe: L-leucyl-L-leucine methyl ester; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MAPK: mitogen-activated protein kinase; MITF: melanocyte inducing transcription factor; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ncRNA: non-coding RNA; RNP: ribonucleoprotein; SF: sorafenib; SQSTM1/p62: sequestosome 1; STS: staurosporine; tdRs: tRNA-derived RNAs; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcription factor EB; vtRNA: vault RNA transcript.

小非编码RNA <i>VTRNA1-1</i>（穹窿RNA1-1，vault RNA 1–1）已知可在多种恶性细胞系中赋予细胞凋亡抵抗能力，并可调控肝细胞中的巨自噬/自噬流（macroautophagic/autophagic flux），由此凸显其促存活作用。本研究揭示了<i>VTRNA1-1</i>的全新功能：其可调控<i>体外</i>及<i>体内</i>肿瘤细胞增殖、肿瘤发生及化疗耐药性。在人肝细胞癌细胞中敲除<i>VTRNA1-1</i>后，转录因子EB（transcription factor EB，TFEB）的核定位水平降低，进而导致协同溶酶体表达与调控（coordinated lysosomal expression and regulation，CLEAR）网络基因的表达下调，以及溶酶体区室功能异常。本研究进一步证实，因<i>VTRNA1-1</i>缺失导致的溶酶体功能受损，可增强常规化疗药物的抗肿瘤效应。最后，<i>VTRNA1-1</i>的缺失会降低药物的溶酶体趋向性，使细胞内药物化合物的生物利用度提升，由此显著抑制<i>体外</i>及<i>体内</i>的肿瘤细胞增殖。上述研究结果揭示了<i>VTRNA1-1</i>在细胞内分解代谢区室中此前未被发现的功能，并阐明了其在溶酶体介导的化疗耐药中的作用。**缩写说明：** ATP6V0D2：ATP酶H+转运V0亚基d2（ATPase H+ transporting V0 subunit d2）；BafA：巴弗洛霉素A1（bafilomycin A1）；CLEAR：协同溶酶体表达与调控网络（coordinated lysosomal expression and regulation）；CQ：氯喹（chloroquine）；DMSO：二甲基亚砜（dimethyl sulfoxide）；GST-BHMT：谷胱甘肽S-转移酶N端连接甜菜碱-同型半胱氨酸S-甲基转移酶（glutathionine S-transferase N-terminal to betaine–homocysteine S-methyltransferase）；HCC：肝细胞癌（hepatocellular carcinoma）；LAMP1：溶酶体相关膜蛋白1（lysosomal associated membrane protein 1）；LLOMe：L-亮氨酰-L-亮氨酸甲酯（L-leucyl-L-leucine methyl ester）；MAP1LC3B/LC3：微管相关蛋白1轻链3β（microtubule associated protein 1 light chain 3 beta）；MAPK：丝裂原活化蛋白激酶（mitogen-activated protein kinase）；MITF：黑素细胞诱导转录因子（melanocyte inducing transcription factor）；MTT：3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide）；ncRNA：非编码RNA（non-coding RNA）；RNP：核糖核蛋白（ribonucleoprotein）；SF：索拉非尼（sorafenib）；SQSTM1/p62：自噬受体p62/隔离体1（sequestosome 1）；STS：星形孢菌素（staurosporine）；tdRs：tRNA来源RNA（tRNA-derived RNAs）；TFE3：结合IGHM增强子3的转录因子（transcription factor binding to IGHM enhancer 3）；TFEB：转录因子EB（transcription factor EB）；vtRNA：穹窿RNA转录本（vault RNA transcript）。