Increase,miRNA levels leads to Decrease,SIRT1(sirtuin 1) levels

There are several signaling pathways that establishing the role of increased miRNA expression in downregulating SIRT 1 gene few of which are listed as follows;

Butyrate–miR-22–SIRT1

Butyrate, a short-chain fatty acid, is produced by the intestinal microbiome via anaerobic fermentation and is subsequently absorbed by the hepatocytes (Besten et al., 2013). Butyrate has been demonstrated to cause apoptosis and reduce carcinogenesis in a variety of cancers (Tailor et al.,2014; Rahmani et al.,2002). Although butyrate has been shown to suppress SIRT1 gene expression in various cancers, this has yet to be proven in hepatocellular carcinoma (HCC) (Iglesias et al., 2007). In HCC, miR-22 was found to be downregulated, and its low levels aided carcinogenesis (Zhang et al.,2010). The Huh7 cells' in vitro proliferation was decreased by miR-22 expression, which activated apoptosis. In Huh7 cells, on the other hand, SIRT1 expression was high, which enhanced the expression of antioxidants such superoxide dismutase (SOD), allowing cell growth to continue (Chen et al.,2012). Butyrate upregulated miR-22 in Huh7 cells, which binds directly to the 3′UTR region of SIRT1 and suppresses its expression; this decreased SOD function and increased ROS generation, increasing caspase 3 and cytochrome c activity, and encouraging apoptosis (Pant et al .,2017).Furthermore, by downregulating SIRT1, miR22 increased PTEN and gsk-3 expression and downregulated β catenin and p-akt expression and thus may promote apoptosis and decrease HCC proliferation (Pant et al .,2017).

Notch3–SIRT1–LSD1–SOX2 Signaling Pathway

Lysine demethylase 1 (LSD1) is an epigenetic regulator responsible for demethylating various histones and controls the pluripotency of stem cells (Adamo et al.,2011; Whyte et al., 2012; Thambyrajah et al., 2016).

In comparison to normal hepatic parenchyma, HCC cells overexpress LSD1. Furthermore, LSD1 is highly expressed in LCSCs, where it regulates SOX2 gene transcription, promotes self-renewal and carcinogenesis, and is linked to a poor patient prognosis (Liu et al.,2018). LSD1 demethylated the SOX2 promoter, increasing its expression and improving LCSC stemness in a similar way to SIRT1 via DNMT3A. Acetylation suppresses LSD1's enzymatic activity and promotes its breakdown via UPP. SIRT1 enhanced the stability of LSD1 by deacetylating it . Notch signalling is essential for cell survival and proliferation (Bouras et al., 2008). Notch receptors are overexpressed in most HCCs, and their ligand expression has been linked to aggressive tumour characteristics (Tschaharganeh et al., 2013). The Wnt/-catenin pathway was activated by Notch, which enhanced HCC growth and metastasis (Wang et al.,2016; Wu et al .,2017). Notch signalling has also been demonstrated to increase CSC self-renewal. SIRT1 expression was enhanced by Notch3 signalling, which also promoted LDS1 deacetylation and activated LSD1 which consequently promotes LCSC self-renewal. The Notch3- dependent pathway was crucial for LCSC self-renewal and in vivo tumor dissemination.

MiR-133b is a tumour suppressor that has been found to be significantly decreased in a variety of malignancies (Hu et al.,2010). When compared to paired neighbouring normal tissue, miR-133b expression was shown to be lower in the majority of HCC samples (El-Halawany et al.,2015). Furthermore, overexpression of miR-133b in HepG2 cells inhibited HCC cell growth and invasion while promoting apoptosis (Tian et al.,2016). In nude mice with orthotopic HepG2 cell tumours, increase of miR-133b also reduced tumour growth. In human HCC cells, miR-133b targets SIRT1 and has an adverse relationship with it. Increased miR-133b expression significantly reduced SIRT1 mRNA and protein expression. Overall, miR-133b appears to have an anti-cancer effect in HCC cells through suppressing SIRT1 expression.

In Huh7 and HepG2 cell lines, inhibiting the SIRT1–SREBP pathway lowered proliferation and DNA synthesis, reduced lipid anabolism, and repressed tumorigenesis (Zhang et al.,2014).

MiR-486 inhibits HCC invasion and tumorigenicity by directly targeting and suppressing SIRT1 expression. This reduced the tumorigenic and chemo-resistant features of LCSCs and inhibited HCC invasion and tumorigenicity (Yan et al.,2019).

The miR-29 family inhibits tumour growth by targeting Mcl-1 and Bcl-2. MiR-29c inhibits hepatocytic SIRT1 and so has tumor-suppressing properties. Ectopic miR-29c expression suppressed cell growth by lowering SIRT1 expression. In hepatocytes, miR-29c directly targets and inhibits SIRT1 mRNA translation (Bae et al., 2014).

SIRT1 was downregulated at the mRNA and protein levels when miR-138 expression was increased. MiR-138 binds to the SIRT1 gene's 3′UTR unique complimentary site and inhibits SIRT1 expression directly, preventing HCC proliferation, migration, and invasion (Luo et al.,2017).When compared to the normal hepatic cell line L02, SIRT1 is overexpressed, while miR-138 levels are lowered in HepG2, SMMC7721, Bel7404, and HCCM3

• In Jiang et al's study, the cellular function and molecular mechanism of miR2045p in hepatocellular cancer were investigated (HCC)(Jiang et al.,2016). Real-time reverse transcription polymerase chain reaction was used to analyze at SIRT1 mRNA and miR2045p. Western blotting was used to determine SIRT1 protein levels. To confirm colony formation, a cell proliferation assay was used. The invasion experiment was carried out using a transwell technique.

-Overexpression of miR2045p in human HCC cell lines (BEL7405 and QGY7701) resulted in cell death, increased apoptosis, and increased drug sensitivity. SIRT1 levels were inversely associated to miR2045p levels and were overexpressed in human HCC tissues. These findings suggest that miR204 5p and SIRT1 may play a role in the progression of HCC.

-Introducing miR20405p into BEL7405 and QGY7701 cells lowered SIRT1 mRNA and protein expression. These findings imply that in HCC cells, SIRT1 is a direct target of miR2045.

-It was also found that SIRT1 was negatively related to miR‐204‐5p expression in HCC tissues.

• Using real-time PCR and western blot assays, Luo et al discovered that miR-138 expression was low while sirtuin type 1 (Sirt1) mRNA expression was high in hepatocellular carcinoma tissues and cell lines, and that miR-138 functions were achieved by targeting Sirt1 using luciferase reporter gene vector and RNA immunoprecipitation assays(Luo et al .,2017).

-Using CCK-8 and BrdU tests, overexpression of miR-138 reduced Sirt1 expression and hindered cell growth.

-Forced production of Sirt1 in cells could partially reverse the inhibitory impact of miR-138. The study findings demonstrated that miR-138 plays a critical role in the regulation of hepatocellular carcinoma cell development via the miR-138/Sirt1 axis, and that miR-138 could be an important future target for hepatocellular carcinoma clinical therapy.

• Shen et al showed that downregulation of miR-199b is associated with distant metastasis in colorectal cancer via activation of SIRT1 and inhibition of CREB/KISS1 signalling(Shen et al., 2016).
• MiR-199b expression levels in six CRC cell lines in comparison to NCM460, a normal colorectal cell line. qRT-PCR detection of relative miR-199b expression after transfection with miR-199b mimics and its negative control (NC).After overexpression of miR-199b, the invasive capacity of SW480 and SW620 cells was examined using the Transwell test.The migratory ability of SW620 and SW480 cells was assessed using a wound healing test. Quantification was done by measuring the wound's smallest clearance distance.After overexpression of miR-199b, Western blot examination revealed the expression levels of invasion-related molecules MMP2 and MMP9, the epithelial-mesenchymal transition (EMT) marker E-cadherin, and Vimentin.The expression of miR-199b was shown to be adversely linked with SIRT1 in CRC specimens in the study. The effects of SIRT1 knockdown on biological behaviour were similar to those of miR-199b overexpression.Furthermore,  Human Tumor Metastasis PCR Array revealed that KISS1 was one of SIRT1's downstream targets. SIRT1 silencing increased the acetylation of the transcription factor CREB, which increased KISS1 expression. The latter was activated further by attaching to the KISS1 promoter, which resulted in transcription.

-According to the findings, miR-199b modulates the SIRT1/CREB/KISS1 signalling pathway and could be used as a prognostic marker or a novel treatment target for CRC patients.

• A study by Tian et al found that MicroRNA-133b targets Sirt1 and suppresses hepatocellular carcinoma cell progression(Tian et al., 2016).qRT-PCR was used to examine miR-133b expression levels in 37 cases of primary hepatoma carcinoma tissues and their surrounding normal equivalents.Sirt1 is a direct target of miR-133b, and its expression in HCC is inversely linked with that of miR-133b.After transfecting the miR-133b expression plasmid into HepG2 and SMMC7721 cells, Sirt1 expression was elevated, indicating that the effect of miR-133b on HCC cells is dependent on Sirt1 repression. In comparison to the control group of HCC cells pretreated with miR-133b overexpression vector, transfection of the Sirt1 overexpression vector restored Sirt1 protein levels.Restoring Sirt1 partially reverses the effect of up-regulation of miR-113b on HCC cell proliferation, invasion, and apoptosis in vitro.
• In liver cancer, Yan et al discovered that MicroRNA 486 5p acts as a tumour suppressor of proliferation and cancer stem-like cell characteristics by targeting Sirt1(Yan et al.,2019).

-The qPCR analysis revealed that miR 486 decreased Sirt1 expression in HepG2 and PLC cells. Transfection of a miR 486 inhibitor, on the other hand, enhanced Sirt1 mRNA levels.

-The study focused primarily on Sirt1 as a miR 486 target gene. A luciferase assay in 293T cells was used to further investigate the connection between miR 486 and Sirt1 3'UTR. The Dual Luciferase Assay System was used to detect luciferase activity in cells co transfected with miR 486 mimics or NC using the pGL3-Sirt1-3'UTR vector or pGL3 basic vector. Sirt1-3'UTR reporter firefly luciferase activity was considerably reduced by co-transfection with miR 486 mimics, but not by the pGL3 reporter. Western blot analysis revealed that in the miR 486 overexpression group, Sirt1 was downregulated. Furthermore, IHC labelling revealed that Sirt1 expression was downregulated in tumour tissues produced from miR 486 overexpression cells.

• Zhang et al reported that MicroRNA-22 functions as a tumor suppressor by targeting SIRT1 in renal cell carcinoma (Zhang et al., 2016).

-Real-time quantitative RT-PCR was used to determine the miR-22 expression pattern (qRT-PCR).

-Quantitative real-time PCR results revealed that miR-22 was considerably downregulated in RCC samples compared to non-cancerous tissues, and this was related with tumour stage and lymph node metastasis.In vitro, forced overexpression of miR-22 decreased proliferation, migration, and invasion, as well as promoted cell death, and suppressed tumour growth in vivo, according to a functional investigation.In addition, a luciferase reporter test showed SIRT1 as a direct target of miR-22.

-MiR-22 overexpression suppressed epithelial-mesenchymal transition via activating p53 and its downstream targets p21 and PUMA, as well as the apoptosis markers CASP3 and PARP (EMT). These findings revealed that miR-22 acted as a tumour suppressor in RCC and inhibited RCC growth and metastasis by directly targeting SIRT1, implying an unique therapeutic role for RCC treatment.

• Astragalus Polysaccharides(APS) inhibits Tumorigenesis and Lipid Metabolism Through the miR-138-5p/SIRT1/SREBP1 Pathway in Prostate Cancer ( Guo et al.,2020).APS was discovered to suppress the proliferation and invasion of PCa cells in vitro and in vivo in a dose- and time-dependent manner in the current investigation.Under APS exposure, SIRT1 expression was significantly decreased, according to microarray results.

-SIRT1 knockdown enhances AMPK/SREBP1 signalling and its related target genes considerably.

• Bae et al reported that MicroRNA-29c functions as a tumor suppressor by direct targeting oncogenic SIRT1 in hepatocellular carcinoma(Bae et al., 2014).

-In this study, SIRT1 expression in a subset of HCCs was performed.

-The study found that overexpression of SIRT1 increased HCC cell proliferation by inactivating p21Cip1, p27Kip1, and p15INK4B, as well as activating CDK2, CDK6, cyclin D3, and cyclin D1.

-In addition, miRNA expression profiling was used to explore for deregulated miRNAs in HCC, and five miRNAs targeting SIRT1 were shown to be highly downregulated in the disease.

-The liver cancer cell lines SNU-182, HepG2, and Hep3B grew slower when SIRT1 was knocked off.

-miR-29c inhibits SIRT1 mRNA translation in the liver, acting as a tumour suppressor. In the rescue experiment, SIRT1-expressing plasmid (pcDNA3.1- SIRT1-His) or Mock (empty vector, pcDNA3.1-His) were introduced into SNU-182 cell lines in the presence or absence of ectopic miR-29c expression. MiR-29c mimic (miR-29c) and 30 UTR-negative SIRT1 expression plasmids were transfected into cells (pcDNA3.1-SIRT1-His).

-After 48 h of transfection, endogenous SIRT1, not ectopically expressed SIRT1 (SIRT1-His), was suppressed by ectopic miR-29c transfection as found in Western blot analysis .

-Anti-growth effect of ectopic miR-29c was attenuated by co-transfection of SIRT1-expressing plasmid

• In a study by Zhou et al,the miR-34a overexpression vector or scramble control was transfected into human Hep3B and Huh7 cell lines(Zhou et al 2017).

-To determine the impact of miR-34a expression on HCC cell invasion and migration, researchers performed Transwell assays, Matrigel, and wound healing experiments.

-Using quantitative reverse transcription polymerase chain reaction, the expression of miR-34a and the mRNA expression of other related proteins was discovered, and protein levels were determined using western blot analysis.

-MiR-34a expression was considerably downregulated in Hep3B and Huh7 cells compared to the control, however this was reversed by transfection with exogenous miR-34a.

-Overexpression of miR-34a increased the expression of sirtuin 1 while decreasing the amount of acetylate-p53.

• Study by Fu et al concluded that MiRNA-200a induce cell apoptosis in renal cell carcinoma by directly targeting SIRT1(Fu et al.,2018).The expression of miR-200a was found to be considerably lower in renal cell carcinoma (RCC) specimens and RCC cell lines in the study. In RCC cell lines, restoring miR-200a decreased cell growth, halted cell cycle progression, and accelerated cell death.

-Sirtuin 1 (SIRT1) was identified as one of the downregulated proteins after miR-200a overexpression in 786-O cells using qRT-PCR array technique. SIRT1 was confirmed as a direct target of miR-200a after a second assay using a luciferase reporter system. Furthermore, knocking down SIRT1 with siRNA could partially mimic the effects of miR-200a overexpression.

-Overexpression of truncated SIRT1 (without an endogenous 30 -UTR) on 786-O cells, on the other hand, may rescue the effect of miR-200a overexpression, suggesting that the SIRT1 30 -UTR is preferentially targeted by miR-200a.

-These findings add to the growing body of data for the miR-200a's tumor-suppressive effect in RCC, as well as establishing a novel regulatory mechanism that could play a role in SIRT1 overexpression in RCC.

• Study by Lian et al  performed Quantitative real-time PCR analysis  to detect the expression of microRNA-128 (miR-128) in tissues from patients with CRC and CRC cell lines. The effect of miR-128 on Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), an anti-tumor medication, caused cytotoxicity against CRC cell lines was assessed using MTT assays (Lian et al.,2018).

-Flow cytometry was used to detect the distribution of death receptor 5 (DR5) and the formation of reactive oxygen species (ROS).Western blot, flow cytometry, and luciferase reporter assays were used to look into the mechanism and pathway of miR-128-induced apoptosis in TRAIL-treated CRC cells.

-MiR-128 expression was found to be downregulated in CRC tumour tissues as well as CRC cell lines in vitro. MiR-128 overexpression made CRC cells more susceptible to TRAIL-induced cytotoxicity through triggering apoptosis. MiR-128 directly targeted sirtuin 1 (SIRT1) in CRC cells, according to bioinformatics, western blot analysis, and luciferase reporter tests. Overexpression of miR128 reduced SIRT1 expression, which increased ROS generation in TRAIL-treated CRC cells.

This increase of ROS subsequently induced DR5 expression, and thus increased TRAIL-induced apoptosis in CRC cells.

• A study by Guan et al confirmed SIRT1 as a direct target of miR-30a by measuring SIRT1 expression in lung cancer cells following overexpression or knockdown of miR-30a and using a luciferase assay (Guan et al.,2017).

-In vitro and in vivo, miR-30a decreased lung cancer cell proliferation, invasion, and increased apoptosis through inhibiting SIRT1.This research discovered a new regulatory axis in which miR-30a and SIRT1 control lung cancer cell proliferation, invasion, and apoptosis, as well as lung carcinogenesis. After overexpression or knockdown of miR-30a, two human lung cancer cell lines (A549 and H1975) were employed to confirm the direct link between miR-30a and SIRT1.Cellular levels of miR-30a were greatly elevated in A549 and H1975 cells transfected with miR-30a mimics and dramatically decreased in cells transfected with miR-30a inhibitor, as expected.As a result, overexpression of miR-30a dramatically decreased SIRT1 protein expression in A549 and H1975 cells, whereas the miR-30a inhibitor considerably boosted SIRT1 protein levels in lung cancer cells. The studies were repeated, and the expression of SIRT1 mRNA after transfection was evaluated to see how much miR-30a influenced SIRT1 expression. The level of SIRT1 mRNA was unaffected by overexpression or knockdown of miR-30a.

              - In cells co-transfected with luciferase reporter plasmid and miR-30a mimics, luciferase activity was drastically reduced. Then, to delete the expected miR-30a binding site, point mutations were induced into the SIRT1 3′-UTR binding region. The overexpression or knockdown of miR30a has no effect on this mutant luciferase reporter. This finding revealed that the binding sites play a significant role in miR-30a-SIRT1 mRNA interaction.

• Yang et al suggested Down-Regulation of miR-221 and miR-222 Restrain Prostate Cancer Cell Proliferation and Migration That Is Partly Mediated by Activation of SIRT1(Yang et al.,2014).

- When compared to LNCap cells, PC-3 cells had higher levels of miR-221 and miR-222 expression. The proliferation and migration rates of PC-3 cells dropped after miR-221 or miR-222 expression was inhibited, but the apoptosis rate rose. Furthermore, after cells were transfected with miR-221 or miR-222 inhibitors, SIRT1 protein was up-regulated. In comparison to the controls, cells transfected with siSIRT1 demonstrated greater migration and a lower rate of apoptosis, but no significant influence on cell proliferation. Although there was a negative connection between miR-221 and SIRT1, no direct target relationship was discovered. These findings show that miR-221 and miR-222 are abundant in PC-3 cells. In prostate cancer cells, inhibiting these proteins reduces cell proliferation and migration while increasing apoptosis. Upregulation of SIRT1 may be responsible for these effects.

Not specific.

No specific pattern of response response relationship was observed.

In study by Jiang et al,  it was shown that miR‐204‐5p targeting SIRT1 regulates hepatocellular carcinoma progression. The results were noted within 48 hours during the experiment (Jiang et al., 2016).

p53-miR-34a–SIRT1 Signaling Pathway

0404 is a DNA-damaging substance that has no cytotoxic effects on human hepatocytes that aren't malignant. In an in vivo HepG2 HCC model, 0404 caused apoptosis and inhibited proliferation. P53 WT HepG2 cells, on the other hand, were more susceptible to 0404 than p53 mutant Huh7 cell lines (Xia et al.,2017). P53 influences the expression of several miRs. As a result, a large number of miRs target the 3′UTR region of the p53 mRNA. As a result, p53 and miRs could establish a feedback loop (Zhang et al., 2015). The miR-34 family has been identified as the most common p53-induced miRs and is commonly suppressed in diverse malignancies (Xiao et al.,2014, Lou et al.,2015). In HCC cells, miR-34a increased p53 transcription and acetylation while also inducing apoptosis. 0404 enhanced p53 and miR-34a expression, elevated acetylated p53, and downregulated SIRT1 protein expression in HepG2 but not Huh7 cell lines, inhibiting HCC growth (Xiao et al.,2014).

In HCC, the lncRNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) is highly expressed, promoting development and invasion. MALAT1 stimulates the formation of HCC CSCs by activating the mechanistic target of rapamycin (mTOR) signalling pathway (Malakar et al.,2017;Yuan et al 2016). MiR-204, in contrast to MALAT1, promotes apoptosis by activating p53 and suppressing Bcl-2, an anti-apoptotic protein (Ryan et al.,2012 ). Cancer stemness and EMT were also suppressed by miR-204, which increased chemosensitivity (Ryan et al.,2012 ;Sacconi et al.,2012). MALAT1 expression, on the other hand, was negatively linked with miR-204 levels. MALAT1 binds to miR-204 and inhibits its expression by binding directly to it (Hou et al., 2017). SIRT1 appears to play a key role in the interaction between MALAT1 and miR-204. SIRT1 is recognised to play a role in HCC EMT, migration, and invasion. MiR-204 specifically targets SIRT1 and silences it (Hou et al.,2017).

However, because SIRT1 and MALAT1 bind to the same miR-204 region, MALAT1 may compete with SIRT1 for miR204 binding, reducing miR-204-induced SIRT1 suppression. Overall, MALAT1 inhibited miR-204 activity, resulting in an elevation in SIRT1, which encouraged HCC migration and invasion (Hou et al., 2017). MALAT1 inhibition reduced the aggressiveness of HCC, making it a possible therapeutic target (Hou et al., 2017).